Detection of HIV

ABSTRACT

The present invention related to oligonucleotides for use in amplifying and detecting HIV nucleic acid in a sample.

This application is a continuation-in-part of application Ser. No.08/469,067, filed Jun. 6, 1995, now U.S. Pat. No. 5,824,518, which is acontinuation of application Ser. No. 07/550,837, filed Jul. 10, 1990,now U.S. Pat. No. 5,480,784, which is a continuation-in-part ofapplication Ser. No. 07/379,501, filed Jul. 11, 1989, now abandoned,with application Ser. Nos. 08/469,067 and 07/550,837 being herebyincorporated by reference herein in their entirety (including thedrawings).

FIELD OF THE INVENTION

This invention relates to methods for increasing the number of copies ofa specific nucleic acid sequence or “target sequence” which may bepresent either alone or as a component, large or small, of a homogeneousor heterogeneous mixture of nucleic acids. The mixture of nucleic acidsmay be that found in a sample taken for diagnostic testing,environmental testing, for research studies, for the preparation ofreagents or materials for other processes such as cloning, or for otherpurposes.

The selective amplification of specific nucleic acid sequences is ofvalue in increasing the sensitivity of diagnostic and environmentalassays while maintaining specificity; increasing the sensitivity,convenience, accuracy and reliability of a variety of researchprocedures; and providing ample supplies of specific oligonucleotidesfor various purposes.

The present invention is particularly suitable for use in environmentaland diagnostic testing due to the convenience with which it may bepracticed.

BACKGROUND OF THE INVENTION

The detection and/or quantitation of specific nucleic acid sequences isan increasingly important technique for identifying and classifyingmicroorganisms, diagnosing infectious diseases, detecting andcharacterizing genetic abnormalities, identifying genetic changesassociated with cancer, studying genetic susceptibility to disease, andmeasuring response to various types of treatment. Such procedures havealso found expanding uses in detecting and quantitating microorganismsin foodstuffs, environmental samples, seed stocks, and other types ofmaterial where the presence of specific microorganisms may need to bemonitored. Other applications are found in the forensic sciences,anthropology, archaeology, and biology where measurement of therelatedness of nucleic acid sequences has been used to identify criminalsuspects, resolve paternity disputes, construct genealogical andphylogenetic trees, and aid in classifying a variety of life forms.

A common method for detecting and quantitating specific nucleic acidsequences is nucleic acid hybridization. This method is based on theability of two nucleic acid strands which contain complementary oressentially complementary sequences to specifically associate, underappropriate conditions, to form a double-stranded structure. To detectand/or quantitate a specific nucleic acid sequence (known as the “targetsequence”), a labelled oligonucleotide (known as a “probe”) is preparedwhich contains sequences complementary to those of the target sequence.The probe is mixed with a sample suspected of containing the targetsequence, and conditions suitable for hybrid formation are created. Theprobe hybridizes to the target sequence if it is present in the sample.The probe-target hybrids are then separated from the single-strandedprobe in one of a variety of ways. The amount of label associated withthe hybrids is measured.

The sensitivity of nucleic acid hybridization assays is limitedprimarily by the specific activity of the probe, the rate and extent ofthe hybridization reaction, the performance of the method for separatinghybridized and unhybridized probe, and the sensitivity with which thelabel can be detected. Under the best conditions, direct hybridizationmethods such as that described above can detect about 1×10⁵ to 1×10⁶target molecules. The most sensitive procedures may lack many of thefeatures required for routine clinical and environmental testing such asspeed, convenience, and economy. Furthermore, their sensitivities maynot be sufficient for many desired applications. Infectious diseases maybe associated with as few as one pathogenic microorganism per 10 ml ofblood or other specimen. Forensic investigators may have available onlytrace amounts of tissue available from a crime scene. Researchers mayneed to detect and/or quantitate a specific gene sequence that ispresent as only a tiny fraction of all the sequences present in anorganism's genetic material or in the messenger RNA population of agroup of cells.

As a result of the interactions among the various components andcomponent steps of this type of assay, there is almost always an inverserelationship between sensitivity and specificity. Thus, steps taken toincrease the sensitivity of the assay (such as increasing the specificactivity of the probe) may result in a higher percentage of falsepositive test results. The linkage between sensitivity and specificityhas been a significant barrier to improving the sensitivity ofhybridization assays. One solution to this problem would be tospecifically increase the amount of target sequence present using anamplification procedure. Amplification of a unique portion of the targetsequence without requiring amplification of a significant portion of theinformation encoded in the remaining sequences of the sample could givean increase in sensitivity while at the same time not compromisingspecificity. For example, a nucleic acid sequence of 25 bases in lengthhas a probability of occurring by chance of 1 in 4²⁵ or 1 in 10¹⁵ sinceeach of the 25 positions in the sequence may be occupied by one of fourdifferent nucleotides.

A method for specifically amplifying nucleic acid sequences termed the“polymerase chain reaction” or “PCR” has been described by Mullis et al.(See U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 and Europeanpatent applications 86302298.4, 86302299.2, and 87300203.4 and Methodsin Enzymology, Volume 155, 1987, pp. 335-350). The procedure usesrepeated cycles of primer-dependent nucleic acid synthesis occurringsimultaneously using each strand of a complementary sequence as atemplate. The sequence which is amplified is defined by the locations ofthe primer molecules that initiate synthesis. The primers arecomplementary to the 3′-terminal portion of the target sequence or itscomplement and must complex with those sites in order for nucleic acidsynthesis to begin. After extension product synthesis, the strands areseparated, generally by thermal denaturation, before the next synthesisstep. In the PCR procedure, copies of both strands of a complementarysequence are synthesized.

The strand separation step used in PCR to separate the newly synthesizedstrands at the conclusion of each cycle of the PCR reaction is oftenthermal denaturation. As a result, either a thermostable enzyme isrequired or new enzyme must be added between thermal denaturation stepsand the initiation of the next cycle of DNA synthesis. The requirementof repeated cycling of reaction temperature between several differentand extreme temperatures is a disadvantage of the PCR procedure. Inorder to make the PCR convenient, expensive programmable thermal cyclinginstruments are required.

The PCR procedure has been coupled to RNA transcription by incorporatinga promoter sequence into one of the primers used in the PCR reaction andthen, after amplification by the PCR procedure for several cycles, usingthe double-stranded DNA as template for the transcription ofsingle-stranded RNA. (See, e.g. Murakawa et al., DNA 7:287-295 (1988).

Other methods for amplification of a specific nucleic acid sequencecomprise a series of primer hybridization, extending and denaturingsteps to provide an intermediate double stranded DNA molecule containinga promoter sequence through the use of a primer. The double stranded DNAis used to produce multiple RNA copies of the target sequence. Theresulting RNA copies can be used as target sequences to produce furthercopies and multiple cycles can be performed. (See, e.g., Burg, et al.,WO 89/1050 and Gingeras, et al., WO 88/10315.)

Methods for chemically synthesizing relatively large amounts of DNA of aspecified sequence in vitro are well known to those skilled In the art;production of DNA in this way is now commonplace. However, theseprocedures are time-consuming and cannot be easily used to synthesizeoligonucleotides much greater in length than about 100 bases. Also, theentire base sequence of the DNA to be synthesized must be known. Thesemethods require an expensive instrument capable of synthesizing only asingle sequence at one time. Operation of this instrument requiresconsiderable training and expertise. Methods for the chemical synthesisof RNA have been more difficult to develop.

Nucleic acids may be synthesized by techniques which involve cloning orinsertion of specific nucleic acid sequences into the genetic materialof microorganisms so that the inserted sequences are replicated when theorganism replicates. If the sequences are inserted next to anddownstream from a suitable promoter sequence, RNA copies of the sequenceor protein products encoded by the sequence may be produced. Althoughcloning allows the production of virtually unlimited amounts of specificnucleic acid sequences, due to the number of manipulations involved itmay not be suitable for use in diagnostic, environmental, or forensictesting. Use of cloning techniques requires considerable training andexpertise. The cloning of a single sequence may consume severalman-months of effort or more.

Relatively large amounts of certain RNAs may be made using a recombinantsingle-stranded-RNA molecule having a recognition sequence for thebinding of an RNA-directed polymerase, preferably Qβ replicase. (See,e.g., U.S. Pat. No. 4,786,600 to Kramer, et al.) A number of steps arerequired to insert the specific sequence into a DNA copy of the variantmolecule, clone it into an expression vector, transcribe it into RNA andthen replicate it with Qβ replicase.

SUMMARY OF THE INVENTION

The present invention is directed to novel methods of synthesizingmultiple copies of a target nucleic acid sequence which areautocatalytic (i e., able to cycle automatically without the need tomodify reaction conditions such as temperature, pH, or ionic strengthand using the product of one cycle in the next one).

The present method includes (a) treating an RNA target sequence with afirst oligonucleotide which comprises a first primer which has acomplexing sequence sufficiently complementary to the 3′-terminalportion of the target to complex therewith and which optionally has asequence 5′ to the priming sequence which includes a promoter for an RNApolymerase under conditions whereby an oligonucleotide/target sequencecomplex is formed and DNA synthesis may be initiated, (b) extending thefirst primer in an extension reaction using the target as a template togive a first DNA primer extension product complementary to the RNAtarget, (c) separating the DNA extension product from the RNA targetusing an enzyme which selectively degrades the RNA target; (d) treatingthe DNA primer extension product with a second oligonucleotide whichcomprises a primer or a splice template and which has a complexingsequence sufficiently complementary to the 3′-terminal portion of theDNA primer extension product to complex therewith under conditionswhereby an oligonucleotide/target sequence complex is formed and DNAsynthesis may be initiated, provided that if the first oligonucleotidedoes not have a promoter, then the second oligonucleotide is a splicetemplate which has a sequence 5′ to the complexing sequence whichincludes a promoter for an RNA polymerase; (e) extending the 3′-terminusof either the second oligonucleotide or the first primer extensionproduct, or both, in a DNA extension reaction to produce a template forthe RNA polymerase; and (f) using the template to produce multiple RNAcopies of the target sequence using an RNA polymerase which recognizesthe promoter sequence. The oligonucleotide and RNA copies may be used toautocatalytically synthesize multiple copies of the target sequence.

In one aspect of the present invention, the general method includes (a)treating an RNA target sequence with a first oligonucleotide whichcomprises a first primer which has a complexing sequence sufficientlycomplementary to the 3′-terminal portion of the target to complextherewith and which has a sequence 5′ to the complexing sequence whichincludes a promoter for an RNA polymerase under conditions whereby anoligonucleotide/target complex is formed and DNA synthesis may beinitiated, (b) extending the first primer in an extension reaction usingthe target as a template to give a first DNA primer extension productcomplementary to the RNA target, (c) separating the first DNA primerextension product from the RNA target using an enzyme which selectivelydegrades the RNA target; (d) treating the DNA primer extension productwith a second oligonucleotide which comprises a second primer which hasa complexing sequence sufficiently complementary to the 3′-terminalportion of the DNA primer extension product to complex therewith underconditions whereby an oligonucleotide/target complex is formed and DNAsynthesis may be initiated; (e) extending the 3′-terminus of the secondprimer in a DNA extension reaction to give a second DNA primer extensionproduct, thereby producing a template for the RNA polymerase; and (f)using the template to produce multiple RNA copies of the target sequenceusing an RNA polymerase which recognizes the promoter sequence. Theoligonucleotide and RNA copies may be used to autocatalyticallysynthesize multiple copies of the target sequence. This aspect furtherincludes: (g) treating an RNA copy from step (f) with the second primerunder conditions whereby an oligonucleotide/target sequence complex isformed and DNA synthesis may be initiated; (h) extending the 3′ terminusof the second primer in a DNA extension reaction to give a second DNAprimer extension product using the RNA copy as a template; (i)separating the second DNA primer extension product from the RNA copyusing an enzyme which selectively degrades the RNA copy; (j) treatingthe second DNA primer extension product with the first primer underconditions whereby an oligonucleotide/target sequence complex is formedand DNA synthesis may be initiated; (k) extending the 3′ terminus of thesecond primer extension product in a DNA extension reaction to produce atemplate for an RNA polymerase; and (1) using the template of step (k)to produce multiple copies of the target sequence using an RNApolymerase which recognizes the promoter. Using the RNA copies of step(1), steps (g) to (k) may be autocatalytically repeated to synthesizemultiple copies of the target sequence. The first primer which in step(k) acts as a splice template may also be extended in the DNA extensionreaction of step (k).

Another aspect of the general method of the present invention provides amethod which comprises (a) treating an RNA target sequence with a firstprimer which has a complexing sequence sufficiently complementary to the3′ terminal portion of the target sequence to complex therewith underconditions whereby an oligonucleotide/target sequence complex is formedand DNA synthesis may be initiated; (b) extending the 3′ terminus of theprimer in an extension reaction using the target as a template to give aDNA primer extension product complementary to the RNA target; (c)separating the DNA extension product from the RNA target using an enzymewhich selectively degrades the RNA target; (d) treating the DNA primerextension product with a second oligonucleotide which comprises a splicetemplate which has a complexing sequence sufficiently complementary tothe 3′-terminus of the primer extension product to complex therewith anda sequence 5′ to the complexing sequence which includes a promoter foran RNA polymerase under conditions whereby an oligonucleotide/targetsequence complex is formed and DNA synthesis may be initiated; (e)extending the 3′ terminus of the DNA primer extension product to addthereto a sequence complementary to the promoter, thereby producing atemplate for an RNA polymerase; (f) using the template to producemultiple RNA copies of the target sequence using an RNA polymerase whichrecognizes the promoter sequence; and (g) using the RNA copies of step(f), autocatalytically repeating steps (a) to (f) to amplify the targetsequence. Optionally, the splice template of step (d) may also functionas a primer and in step (e) be extended to give a second primerextension product using the first primer extension product as atemplate.

In addition, in another aspect of the present invention, where thesequence sought to be amplified is present as DNA, use of an appropriatePreliminary Procedure generates RNA copies which may then be amplifiedaccording to the General Method of the present invention.

Accordingly, in another aspect, the present invention is directed toPreliminary Procedures for use in conjunction with the amplificationmethod of the present invention which not only can increase the numberof copies present to be amplified, but also can provide RNA copies of aDNA sequence for amplification.

The present invention is directed to methods for increasing the numberof copies of a specific target nucleic acid sequence in a sample. In oneaspect, the present invention involves cooperative action of a DNApolymerase (such as a reverse transcriptase) and a DNA-dependent RNAPolymerase (transcriptase) with an enzymatic hybrid-separation step toproduce products that may themselves be used to produce additionalproduct, thus resulting in an autocatalytic reaction without requiringmanipulation of reaction conditions such as thermal cycling. In someembodiments of the methods of the present invention which include aPreliminary Procedure, all but the initial step(s) of the preliminaryprocedure are carried out at one temperature.

The methods of the present invention may be used as a component ofassays to detect and/or quantitate specific nucleic acid targetsequences in clinical, environmental, forensic, and similar samples orto produce large numbers of copies of DNA and/or RNA of specific targetsequence for a variety of uses. These methods may also be used toproduce multiple DNA copies of a DNA target sequence for cloning or togenerate probes or to produce RNA and DNA copies for sequencing.

In one example of a typical assay, a sample to be amplified is mixedwith a buffer concentrate containing the buffer, salts, magnesium,nucleotide triphosphates, primers and/or splice templates,dithiothreitol, and spermidine. The reaction is then optionallyincubated near 100° C. for two minutes to denature any secondarystructure. After cooling to room temperature, if the target is a DNAtarget without a defined 3′ terminus, reverse transcriptase is added andthe reaction mixture is incubated for 12 minutes at 42° C. The reactionis again denatured near 100° C., this time to separate the primerextension product from the DNA template. After cooling, reversetranscriptase, RNA polymerase, and RNAse H are added and the reaction isincubated for two to four hours at 37° C. The reaction can then beassayed by denaturing the product, adding a probe solution, incubating20 minutes at 60° C., adding a solution to selectively hydrolyze theunhybridized probe, incubating the reaction six minutes at 60° C., andmeasuring the remaining chemiluminescence in a luminometer. (See, e.g.,Arnold, et al., International Publication No. WO 89/02476, publishedMar. 23, 1989, International Application No. PCT/US88/03195, filed Sep.21, 1988, the disclosure of which is incorporated herein by referenceand is referred to as “HPA”). The products of the methods of the presentinvention may be used in many other assay systems known to those skilledin the art.

If the target has a defined 3′ terminus or the target is RNA, a typicalassay includes mixing the target with the buffer concentrate mentionedabove and denaturing any secondary structure. After cooling, reversetranscriptase, RNA polymerase, and RNAse H are added and the mixture isincubated for two to four hours at 37° C. The reaction can then beassayed as described above.

The methods of the present invention and the materials used therein maybe incorporated as part of diagnostic kits for use in diagnosticprocedures.

Definitions

As used herein, the following terms have the following meanings unlessexpressly stated to the contrary.

1. Template

A “template” is a nucleic acid molecule that is being copied by anucleic acid polymerase. A template may be either single-stranded,double-stranded or partially double-stranded, depending on thepolymerase. The synthesized copy is complementary to the template or toat least one strand of a double-stranded or partially double-strandedtemplate. Both RNA and DNA are always synthesized in the 5′ to 3′direction and the two strands of a nucleic acid duplex always arealigned so that the 5′ ends of the two strands are at opposite ends ofthe duplex (and, by necessity, so then are the 3′ ends).

2. Primer, Splice Template

A “primer” is an oligonucleotide that is complementary to a templatewhich complexes (by hydrogen bonding or hybridization) with the templateto give a primer/template complex for initiation of synthesis by a DNApolymerase, and which is extended by the addition of covalently bondedbases linked at its 3′ end which are complementary to the template inthe process of DNA synthesis. The result is a primer extension product.Virtually all DNA polymerases (including reverse transcriptases) thatare known require complexing of an oligonucleotide to a single-strandedtemplate (“priming”) to initiate DNA synthesis, whereas RNA replicationand transcription (copying of RNA from DNA) generally do not require aprimer. Under appropriate circumstances, a primer may act as a splicetemplate as well (see definition of “splice template” that follows).

A “splice template” is an oligonucleotide that complexes with asingle-stranded nucleic acid and is used as a template to extend the 3′terminus of a target nucleic acid to add a specific sequence. The splicetemplate is sufficiently complementary to the 3′ terminus of the targetnucleic acid molecule, which is to be extended, to complex therewith. ADNA- or RNA-dependent DNA polymerase is then used to extend the targetnucleic acid molecule using the sequence 5′ to the complementary regionof the splice template as a template. The extension product of theextended molecule has the specific sequence at its 3′-terminus which iscomplementary to the sequence at the 5′-terminus of the splice template.

If the 3′ terminus of the splice template is not blocked and iscomplementary to the target nucleic acid, it may also act as a primerand be extended by the DNA polymerase using the target nucleic acidmolecule as a template. The 3′ terminus of the splice template can beblocked in a variety of ways, including having a 3′-terminaldideoxynucleotide or a 3′-terminal sequence non-complementary to thetarget, or in other ways well known to those skilled in the art.

Either a primer or a splice template may complex with a single-strandednucleic acid and serve a priming function for a DNA polymerase.

3. Target Nucleic Acid, Target Sequence

A “target nucleic acid” has a “target sequence” to be amplified, and maybe either single-stranded or double-stranded and may include othersequences besides the target sequence which may not be amplified.

The term “target sequence” refers to the particular nucleotide sequenceof the target nucleic acid which is to be amplified. The “targetsequence” includes the complexing sequences to which theoligonucleotides (primers and/or splice template) complex during theprocesses of the present invention. Where the target nucleic acid isoriginally single-stranded, the term “target sequence” will also referto the sequence complementary to the “target sequence” as present in thetarget nucleic acid. Where the “target nucleic acid” is originallydouble-stranded, the term “target sequence” refers to both the (+) and(−) strands.

4. Promoter/Promoter Sequence

A “promoter sequence” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site. For binding, such transcriptases generally require DNAwhich is double-stranded in the portion comprising the promoter sequenceand its complement; the template portion (sequence to be transcribed)need not be double-stranded. Individual DNA-dependent RNA polymerasesrecognize a variety of different promoter sequences which can varymarkedly in their efficiency in promoting transcription. When an RNApolymerase binds to a promoter sequence to initiate transcription, thatpromoter sequence is not part of the sequence transcribed. Thus, the RNAtranscripts produced thereby will not include that sequence.

5. DNA-dependent DNA Polymerase

A “DNA-dependent DNA polymerase” is an enzyme that synthesizes acomplementary DNA copy from a DNA template. Examples are DNA polymeraseI from E. coli and bacteriophage T7 DNA polymerase. All knownDNA-dependent DNA polymerases require a complementary primer to initiatesynthesis. It is known that under suitable conditions a DNA-dependentDNA polymerase may synthesize a complementary DNA copy from an RNAtemplate.

6. DNA-dependent RNA Polymerase (Transcriptase)

A “DNA-dependent RNA polymerase” or “transcriptase” is an enzyme thatsynthesizes multiple RNA copies from a double-stranded orpartially-double stranded DNA molecule having a (usuallydouble-stranded) promoter sequence. The RNA molecules (“transcripts”)are synthesized in the 5′→3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

7. RNA-dependent DNA Polymerase (Reverse Transcriptase)

An “RNA-dependent DNA polymerase” or “reverse transcriptase” is anenzyme that synthesizes a complementary DNA copy from an RNA template.All known reverse transcriptases also have the ability to make acomplementary DNA copy from a DNA template; thus, they are both RNA- andDNA-dependent DNA polymerases. A primer is required to initiatesynthesis with both RNA and DNA templates.

8. RNAse H

An “RNAse H” is an enzyme that degrades the RNA portion of an RNA:DNAduplex. RNAse H's may be endonucleases or exonucleases. Most reversetranscriptase enzymes normally contain an RNAse H activity in additionto their polymerase activity. However, other sources of the RNAse H areavailable without an associated polymerase activity. The degradation mayresult in separation of RNA from a RNA:DNA complex. Alternatively, theRNAse H may simply cut the RNA at various locations such that portionsof the RNA melt off or permit enzymes to unwind portions of the RNA.

9. Plus/Minus Strand(s)

Discussions of nucleic acid synthesis are greatly simplified andclarified by adopting terms to name the two complementary strands of anucleic acid duplex. Traditionally, the strand encoding the sequencesused to produce proteins or structural RNAs was designated as the “plus”strand and its complement the “minus” strand. It is now known that inmany cases, both strands are functional, and the assignment of thedesignation “plus” to one and “minus” to the other must then bearbitrary. Nevertheless, the terms are very useful for designating thesequence orientation of nucleic acids and will be employed herein forthat purpose.

10. Hybridize, Hybridization

The terms “hybridize” and “hybridization” refer to the formation ofcomplexes between nucleotide sequences which are sufficientlycomplementary to form complexes via Watson-Crick base pairing. Where aprimer (or splice template) “hybridizes” with target (template), suchcomplexes (or hybrids) are sufficiently stable to serve the primingfunction required by the DNA polymerase to initiate DNA synthesis.

11. Primer Sequences

The sequences of the primers referred to herein are set forth below.

HBV region 2 primers

(+): 5′CACCAAATGCCCCTATCTTATCAACACTTCCGG3′ (SEQ ID NO:24)

(−): 5′AATTTAATACGACTCACTATAGGGAGACCCGAGATTGAGATCTTCTGCGAC3′ (SEQ IDNO:25)

Probe:

(+): 5′GGTCCCCTAGAAGAAGAACTCCCTCG3′ (SEQ ID NO:23)

HIV region 1 primers

(+): 5′AATTTAATACGACTCACTATAGGGAGACAAGGGACTTTCCGCTGGGGACTTTCC3′ (SEQ IDNO:2)

(−): 5′GTCTAACCAGAGAGACCCAGTACAGGC3′ (SEQ ID NO:3)

Probe sequence:

5′GCAGCTGCTTATATGCAGGATCTGAGGG3′ (SEQ ID NO:1)

HIV region 2 primers

(+): 5′AATTTAATACGACTCACTATAGGGAGACAAATGGCAGTATTCATCCACA3′ (SEQ ID NO:5)

(−): 5′CCCTTCACCTTTCCAGAG3′ (SEQ ID NO:6)

Probe sequence:

(−): 5′CTACTATTCTTTCCCCTGCACTGTACCCC3′ (SEQ ID NO:4)

HIV region 3 primers

(+): 5′CTCGACGCAGGACTCGGCTTGCTG3′ (SEQ ID NO:8)

(−): 5′AATTTAATACGACTCACTATAGGGAGACTCCCCCGCTTAATACTGACGCT3′ (SEQ IDNO:9)

Probe:

(+): 5′GACTAGCGGAGGCTAGAAGGAGAGAGATGGG3′ (SEQ ID NO:7)

HIV region 4 primers

(+): 5′AATTTAATACGACTCACTATAGGGAGAGACCATCAATGAGGAAGCTGCAGAATG3′ (SEQ IDNO:11)

(−): 5′CCATCCTATTTGTTCCTGAAGGGTAC3′ (SEQ ID NO:12)

Probe:

(−): 5′CTTCCCCTTGGTTCTCTCATCTGGCC3′ (SEQ ID NO:10)

HIV region 5 primers

(+): 5′GGCAAATGGTACATCAGGCCATATCACCTAG3′ (SEQ ID NO:14)

(−): 5′AATTTAATACGACTCACTATAGGGAGAGGGGTGGCTCCTTCTGATAATGCTG3′ (SEQ IDNO:15)

Probe:

5′GAAGGCTTTCAGCCCAGAAGTAATACCCATG3′ (SEQ ID NO:13)

BCL-2 chromosomal translocation major breakpoint t(14;18) primers

(−): 5′GAATTAATACGACTCACTATAGGGAGACCTGAGGAGACGGTGACC3′ (SEQ ID NO:41)

(+): 5′TATGGTGGTTTGACCTTTAG3′ (SEQ ID NO:42)

Probes:

5′GGCTTTCTCATGGCTGTCCTTCAG3′ (SEQ ID NO:43)

5′GGTCTTCCTGAAATGCAGTGGTCG3′ (SEQ ID NO:44)

CML chromosomal translocation t(9;22) primers

(−): 5′GAATTAATACGACTCACTATAGGGAGACTCAGACCCTGAGGCTCAAAGTC3′ (SEQ IDNO:45)

(+): 5′GGAGCTGCAGATGCTGACCAAC3′ (SEQ ID NO:46)

Probe:

5′GCAGAGTTCAAAAGCCCTTCAGCGG3′ (SEQ ID NO:31)

12. Specificity

Characteristic of a nucleic acid sequence which describes its ability todistinguish between target and non-target sequences dependent onsequence and assay conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1O depict the General Methods of the present invention.

FIGS. 2A to 2E depict the embodiment of the present invention referredto as Preliminary Procedure I.

FIG. 3 depicts the embodiment of the present invention referred to asPreliminary Procedure II.

FIGS. 4A to 4D depicts the improved amplification method.

FIG. 5 shows the results of experiments testing the hypothesis thatRNAse H from AMV and MMLV and E. coli have specific RNA cleavage sites.

FIG. 6 shows the results of incorporation of ³²P-labeled primers duringamplification.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel methods and compositionsare provided for the amplification of specific nucleic acid targetsequences for use in assays for the detection and/or quantitation ofspecific nucleic acid target sequences or for the production of largenumbers of copies of DNA and/or RNA of specific target sequences for avariety of uses.

I. General Method

In a preferred aspect, the present invention provides an autocatalyticamplification method which synthesizes large numbers of DNA and RNAcopies of an RNA target sequence. The target nucleic acid contains thetarget sequence to be amplified. The target sequence is that region ofthe target nucleic acid which is defined on either end by the primers,splice templates, and/or the natural target nucleic acid termini andincludes both the (+) and (−) strands.

In one aspect, this method comprises treating a target nucleic acidcomprising an RNA target sequence with a first oligonucleotide whichcomprises a first primer which has a complexing sequence sufficientlycomplementary to the 3′-terminal portion of the target sequence tocomplex therewith and which optionally has a sequence 5′ to thecomplexing sequence which includes a promoter sequence for an RNApolymerase under conditions whereby an oligonucleotide/target sequencecomplex is formed and DNA synthesis may be initiated. The firstoligonucleotide primer may also have other sequences 5′ to the primingsequence. The 3′-end of the first primer is extended by an appropriateDNA polymerase in an extension reaction using the RNA as a template togive a first DNA primes extension product which is complementary to theRNA template. The first primer extension product is separated (at leastpartially) from the RNA template using an enzyme which selectivelydegrades the RNA template. Suitable enzymes are those which selectivelyact on the RNA strand of an RNA-DNA complex and include enzymes whichcomprise an RNAse H. Although some reverse transcriptases include anRNAse H activity, it may be preferable to add exogenous RNAse H, such asan E. coli RNAse H.

The single-stranded first primer extension product is treated with asecond oligonucleotide which comprises a second primer or a splicetemplate which has a complexing sequence sufficiently complementary tothe 3′-terminal portion of target sequence contained in the first primerextension product to complex therewith, under conditions whereby anoligonucleotide/target sequence complex is formed and DNA synthesis maybe initiated. If the first primer does not have a promoter then thesecond oligonucleotide is a splice template which has a sequence 5′ tothe complexing region which includes a promoter for an RNA polymerase.Optionally, the splice template may be blocked at its 3′ terminus. The3′ terminus of the second oligonucleotide and/or the primer extensionproduct is extended in a DNA extension reaction to produce a templatefor a RNA polymerase. The RNA copies or transcripts produced mayautocatalytically multiply without further manipulation.

Where an oligonucleotide functions as a splice template, its primerfunction is not required. Thus, the 3′ terminus of the splice templatemay be either blocked or unblocked. The components of the resultingreaction mixture (i.e., an RNA target which allows production of a firstprimer extension product with a defined 3′ terminus, a first primer, anda splice template either blocked or unblocked) function toautocatalytically synthesize large quantities of RNA and DNA.

In one aspect of the present invention, the first and second oligomersboth are primers. The first primer has a sequence 5′ to the complexingsequence which includes a promoter for a RNA polymerase and may includeother sequences. The second primer may also include a sequence 5′ to thecomplexing sequence which may include a promoter for an RNA polymeraseand optionally other sequences. Where both primers have a promotersequence, it is preferred that both sequences are recognized by the sameRNA polymerase unless it is intended to introduce the second promoterfor other purposes, such as cloning. The 3′-end of the second primer isextended by an appropriate DNA polymerase in an extension reaction toproduce a second DNA primer extension product complementary to the firstprimer extension product. Note that as the first primer defined one endof the target sequence, the second primer now defines the other end. Thedouble-stranded product of the second extension reaction is a suitabletemplate for the production of RNA by an RNA polymerase. If the secondprimer also has a promoter sequence, transcripts complementary to bothstrands of the double-stranded template will be produced during theautocatalytic reaction. The RNA transcripts may now have differenttermini than the target nucleic acid, but the sequence between the firstprimer and the second primer remains intact. The RNA transcripts soproduced may automatically recycle in the above system without furthermanipulation. Thus, this reaction is autocatalytic.

If the complexing sequence of the second primer complexes with the 3′terminus of the first primer extension product, the second primer mayact as a splice template and the first primer extension product may beextended to add any sequence of the second primer 5′ to the primingsequence to the first primer extension product. (See, e.g., FIGS. 1E and1G) If the second primer acts as a splice template and includes apromoter sequence 5′ to the complexing sequence, extension of the firstprimer extension product to add the promoter sequence produces anadditional template for an RNA polymerase which may be transcribed toproduce RNA copies of either strand. (See FIGS. 1E and 1G) Inclusion ofpromoters in both primers may enhance the number of copies of the targetsequence synthesized.

Another aspect of the general method of the present invention includesusing a first oligonucleotide which comprises a primer and a secondoligonucleotide which comprises a splice template and which may or maynot be capable of acting as a primer per se (in that it is not itselfextended in a primer extension reaction). This aspect of the generalmethod comprises treating a target nucleic acid comprising an RNA targetsequence with a first oligonucleotide primer which has a complexingsequence sufficiently complementary to the 3′ terminal portion of thetarget sequence to complex therewith under conditions whereby anoligonucleotide/target sequence complex is formed and DNA synthesis maybe initiated. The first primer may have other sequences 5′ to thecomplexing sequence, including a promoter. The 3′ end of the firstprimer is extended by an appropriate DNA polymerase in an extensionreaction using the RNA as a template to give a first primer extensionproduct which is complementary to the RNA template. The first primerextension product is separated from the RNA template using an enzymewhich selectively degrades the RNA template. Suitable enzymes are thosewhich selectively act on the RNA strand of an RNA-DNA complex andinclude enzymes which comprise an RNAse H activity. Although somereverse transcriptases include an RNase H activity, it may be preferableto add exogenous RNAse H, such as an E. coli RNAse H. The singlestranded first primer extension product is treated with a splicetemplate which has a complexing sequence sufficiently complementary tothe 3′-terminus of the primer extension product to complex therewith anda sequence 5′ to the complexing sequence which includes a promoter foran RNA polymerase under conditions whereby an oligonucleotide/targetsequence complex is formed and DNA synthesis may be initiated. The 3′terminus of the splice template may be either blocked (such as byaddition of a dideoxynucleotide) or uncomplementary to the targetnucleic acid (so that it does not function as a primer) or alternativelyunblocked. The 3′ terminus of the first primer extension product isextended using an appropriate DNA polymerase in a DNA extension reactionto add to the 3′ terminus of the first primer extension product asequence complementary to the sequence of the splice template 5′ to thecomplexing sequence which includes the promoter. If the 3′ terminus isunblocked, the splice template may be extended to give a second primerextension product complementary to the first primer extension product.The product of the extension reaction with the splice template (whetherblocked or unblocked) can function as a template for RNA synthesis usingan RNA polymerase which recognizes the promoter. As noted above, RNAtranscripts so produced may automatically recycle in the above systemwithout further manipulation. Thus, the reaction is autocatalytic.

In some embodiments, the target sequence to be amplified is defined atboth ends by the location of specific sequences complementary to theprimers (or splice templates) employed. In other embodiments, the targetsequence is defined at one location of a specific sequence,complementary to a primer molecule employed and, at the opposite end, bythe location of a specific sequence that is cut by a specificrestriction endonuclease, or by other suitable means, which may includea natural 3′ terminus. In other embodiments, the target sequence isdefined at both ends by the location of specific sequences that are cutby one or more specific restriction endonuclease(s).

In a preferred embodiment of the present invention, the RNA targetsequence is determined and then analyzed to determine where RNAse Hdegradation will cause cuts or removal of sections of RNA from theduplex. Analyses can be conducted to determine the effect of the RNAsedegradation of the target sequence by RNAse H present in AMV reversetranscriptase and MMLV reverse transcriptase, by E. coli RNAse H orother sources and by combinations thereof.

In selecting a primer set, it is preferable that one of the primers beselected so that it will hybridize to a section of RNA which issubstantially nondegraded by the RNAse H present in the reactionmixture. If there is substantial degradation, the cuts in the RNA strandin the region of the primer may inhibit initiation of DNA synthesis andprevent extension of the primer. Thus, it is preferred to select aprimer which will hybridize with a sequence of the RNA target, locatedso that when the RNA is subjected to RNAse H, there is no substantialdegradation which would prevent formation of the primer extensionproduct.

The site for hybridization of the promoter-primer is chosen so thatsufficient degradation of the RNA strand occurs to permit removal of theportion of the RNA strand hybridized to the portion of the DNA strand towhich the promoter-primer will hybridize. Typically, only portions ofRNA are removed from the RNA:DNA duplex through RNAse H degradation anda substantial part of the RNA strand remains in the duplex.

Formation of the promoter-containing double stranded product for RNAsynthesis is illustrated in FIG. 4. As illustrated in FIG. 4, the targetRNA strand hybridizes to a primer which is selected to hybridize with aregion of the RNA strand which is not substantially degraded by RNAse Hpresent in the reaction mixture. The primer is then extended to form aDNA strand complementary to the RNA strand. Thereafter, the RNA strandis cut or degraded at various locations by the RNAse H present in thereaction mixture. It is to be understood that this cutting ordegradation can occur at this point or at other times during the courseof the reaction. Then the RNA fragments dissociate from the DNA strandin regions where significant cuts or degradation occur. Thepromoter-primer then hybridizes to the DNA strand at its 3′ end, wherethe RNA strand has been substantially degraded and separated from theDNA strand. Next, the DNA strand is extended to form a double strand DNApromoter sequence, thus forming a template for RNA synthesis. It can beseen that this template contains a double-stranded DNA promotersequence. When this template is treated with RNA polymerase, multiplestrands of RNA are formed.

Although the exact nature of the RNA degradation resulting from theRNAse H is not known, it has been shown that the result of RNAse Hdegradation on the RNA strand of an RNA:DNA hybrid resulted indissociation of small pieces of RNA from the hybrid. It has also beenshown that promoter-primers can be selected which will bind to the DNAafter RNAse H degradation at the area where the small fragments areremoved.

FIGS. 1 and 2, as drawn, do not show the RNA which may remain afterRNAse H degradation. It is to be understood that although these figuresgenerally show complete removal of RNA from the DNA:RNA duplex, underthe preferred conditions only partial removal occurs as illustrated inFIG. 3. By reference to FIG. 1A, it can be seen that the proposedmechanism may not occur if a substantial portion of the RNA strand ofFIG. 1 remains undegraded thus preventing hybridization of the secondprimer or extension of the hybridized second primer to produce a DNAstrand complementary to the promoter sequence. However, based upon theprinciples of synthesis discovered and disclosed in this application,routine modifications can be made by those skilled in the art accordingto the teachings of this invention to provide an effective and efficientprocedure for amplification of RNA.

As may be seen from the descriptions herein and FIGS. 1A to 1O, themethod of the present invention embraces optional variations.

FIG. 1A depicts a method according to the present invention wherein thetarget nucleic acid has additional sequences 3′ to the target sequence.The first oligonucleotide comprises a first primer having a promoter 5′to its complexing sequence which complexes with the 3′ terminal portionof the target sequence of a target nucleic acid (RNA) which hasadditional sequences 3′ to the end of the target sequence. The secondoligonucleotide comprises a second primer which complexes with the 3′terminal portion of the first primer extension product, coinciding withthe 3′ terminus of the first primer extension product. In step (1), thefirst primer does not act as a splice template due to the additionalsequences 3′ to the target sequence; however, in step (10), the firstprimer can act as a splice template, since the second primer extensionproduct does not have additional sequences 3′ to the target sequence.

FIG. 1B depicts a method according to the present invention wherein thetarget nucleic acid (RNA) has additional sequences both 5′ and 3′ to thetarget sequence. The first oligonucleotide is as depicted in FIG. 1A.The second oligonucleotide comprises a primer which complexes to the 3′terminal portion of the target sequence of the first primer extensionproduct which has additional sequences 3′ to the target sequence.

FIG. 1C depicts a target nucleic acid (RNA) which has defined 5′ and 3′ends and, thus, has no additional sequences either 5′ or 3′ to thetarget sequence. The first oligonucleotide is as depicted in FIGS. 1Aand 1B, but since it complexes with the 3′ terminus of the targetnucleic acid, it acts as both a primer and splice template in Step 1.The second oligonucleotide is as depicted in FIG. 1A.

FIG. 1D depicts a target, nucleic acid (RNA) having a defined 3′ endand, thus, has no additional sequences 3′ to the target sequence, butdoes have additional sequences 5′ to the target sequence. The firstoligonucleotide is as depicted in FIG. 1C and functions as both a primerand a splice template. The second oligonucleotide is as depicted in FIG.1B.

FIG. 1E depicts a target nucleic acid (RNA) which has a defined 5′ endbut which has additional sequences 3′ to the target sequence. The firstoligonucleotide is as depicted in FIG. 1A. The second oligonucleotidecomprises a second primer which, since it complexes with the 3′-terminusof the first primer extension product, also comprises a splice template.The second oligonucleotide also has a promoter 5′ to its complexingsequence.

FIG. 1F depicts a target nucleic acid (RNA) having additional sequencesboth 5′ and 3′ to the target sequence. The first oligonucleotide is asdepicted in FIG. 1A. The second oligonucleotide is as depicted in FIG.1E, except it cannot act as a splice template in step (4), since thefirst primer extension product has additional sequences 3′ to the targetsequence.

FIG. 1G depicts a target nucleic acid (RNA) which has both defined 5′and 3′ ends, having no sequences besides the target sequence. The firstoligonucleotide is as depicted in FIG. 1C and the second oligonucleotideas depicted in FIG. 1E. Since the target has no additional sequences,both oligonucleotides also act as splice templates.

FIG. 1H depicts a target nucleic acid (RNA) which has a defined 3′ end,having no sequences 3′ to the target sequence, but has additionalsequences 5′ to the target sequence. The first oligonucleotide is asdepicted in FIGS. 1C and 1G and acts as both a primer and splicetemplate. The second oligonucleotide is as depicted in FIG. 1F.

FIG. 1I depicts a target nucleic acid (RNA) which has a defined 5′terminus and has no additional sequences 5′ to the target sequence, buthas additional sequences 3′ to the target sequence. The firstoligonucleotide comprises a primer without a promoter. The secondoligonucleotide comprises an unblocked splice template which has apromoter 5′ to its completing sequence.

FIG. 1J depicts a target nucleic acid (RNA) which has defined 5′ and 3′terminus and no sequences besides the target sequence. The firstoligonucleotide comprises a primer without a promoter. The secondoligonucleotide is as depicted in FIG. 1I.

FIG. 1K depicts a target nucleic acid (RNA) which has a defined 5′terminus with no additional sequence 5′ to the target sequence, butwhich has additional sequences 3′ to the target sequence. The secondoligonucleotide comprises a splice template having a promoter 5′ to itscompleting sequence, but which is blocked at its 3′ terminus. The secondoligonucleotide is incapable of acting as a primer.

FIG. 1L depicts a target nucleic acid (RNA) which has defined 5′ and 3′ends and no additional sequences besides the target sequence. The firstoligonucleotide acts as both a primer and a splice template. The secondoligonucleotide is a blocked splice template and is as depicted in FIG.1K.

FIG. 1M depicts a target nucleic acid (RNA) which has a defined5′-terminus, and, thus, no additional sequences 5′ to the targetsequence, but which has additional sequences 3′ to the target sequence.The first oligonucleotide is a primer as depicted in FIG. 1I. The secondoligonucleotide is a blocked splice template having a promoter, asdepicted in FIGS. 1K and 1L.

FIG. 1N depicts a target nucleic acid (RNA) which has both defined 5′and 3′ sequences, having no additional sequences besides the targetsequence. The first oligonucleotide comprises a primer without apromoter, as depicted in FIG. 1J. The second oligonucleotide comprises ablocked splice template having a promoter, as depicted in FIGS. 1K, 1Land 1M.

FIG. 1O depicts a target nucleic acid (RNA) which has a defined 5′terminus, having no additional sequences 5′ to the target sequence, butwhich has additional sequences 3′ to the target sequence. Oneoligonucleotide is used for both the first and second oligonucleotide.The oligonucleotide has a 3′-primer sequence which complexes to the3′-terminal portion of the target sequence as shown in Step (1), and hasa 5′ splice template sequence with a promoter which complexes with the3′ terminus of the primer extension product as shown in step (4).

In summary, the methods of the present invention provide a method forautocatalytically synthesizing multiple copies of a target nucleic acidsequence without repetitive manipulation of reaction conditions such astemperature, ionic strength and pH which comprises (a) combining into areaction mixture a target nucleic acid which comprises an RNA targetsequence; two oligonucleotide primers, a first oligonucleotide having acomplexing sequence sufficiently complementary to the 3′ terminalportion of the RNA target sequence (for example the (+) strand) tocomplex therewith and a second oligonucleotide having a complexingsequence sufficiently complementary to the 3′ terminal portion of thetarget sequence of its complement (for example, the (−) strand) tocomplex therewith, wherein the first oligonucleotide comprises a firstprimer which optionally has a sequence 5′ to the complexing sequencewhich includes a promoter and the second oligonucleotide comprises aprimer or a splice template; provided that if the first oligonucleotidedoes not have a promoter, then the second oligonucleotide is a splicetemplate which has a sequence 5′ to the priming sequence which includesa promoter for an RNA polymerase; a reverse transcriptase or RNA and DNAdependent DNA polymerases; an enzyme activity which selectively degradesthe RNA strand of an RNA-DNA complex (such as an RNAse H) and an RNApolymerase which recognizes the promoter. The components of the reactionmixture may be combined stepwise or at once. The reaction mixture isincubated under conditions whereby an oligonucleotide/target sequence isformed, including DNA priming and nucleic acid synthesizing conditions(including ribonucleotide triphosphates and deoxyribonucleotidetriphosphates) for a period of time sufficient whereby multiple copiesof the target sequence are produced. The reaction advantageously takesplace under conditions suitable for maintaining the stability ofreaction components such as the component enzymes and without requiringmodification or manipulation of reaction conditions during the course ofthe amplification reaction. Accordingly, the reaction may take placeunder conditions that are substantially isothermal and includesubstantially constant ionic strength and pH.

The present reaction does not require a denaturation step to separatethe RNA-DNA complex produced by the first DNA extension reaction. Suchsteps require manipulation of reaction conditions such as bysubstantially increasing the temperature of the reaction mixture(generally from ambient temperature to about 80° C. to about 105° C.),reducing its ionic strength (generally by 10× or more) or changing pH(usually increasing pH to 10 or more). Such manipulations of thereaction conditions often deleteriously affect enzyme activities,requiring addition of additional enzyme and also necessitate furthermanipulations of the reaction mixture to return it to conditionssuitable for further nucleic acid synthesis.

Suitable DNA polymerases include reverse transcriptases. Particularlysuitable DNA polymerases include AMV reverse transcriptase and MMLVreverse transcriptase.

Promoters or promoter sequences suitable for incorporation in theprimers and/or splice templates used in the methods of the presentinvention are nucleic acid sequences (either naturally occurring,produced synthetically or a product of a restriction digest) that arespecifically recognized by an RNA polymerase that recognizes and bindsto that sequence and initiates the process of transcription whereby RNAtranscripts are produced. The sequence may optionally include nucleotidebases extending beyond the actual recognition site for the RNApolymerase which may impart added stability or susceptibility todegradation processes or increased transcription efficiency. Promotersequences for which there is a known and available polymerase that iscapable of recognizing the initiation sequence are particularly suitableto be employed. Typical, known and useful promoters include those whichare recognized by certain bacteriophage polymerases such as those frombacteriophage T3, T7 or SP6, or a promoter from E. coli.

Although some of the reverse transcriptases suitable for use in themethods of the present invention have an RNAse H activity, such as AMVreverse transcriptase, it may be preferred to add exogenous RNAse H,such as E. coli RNAse H. Although, as the examples show, the addition ofexogenous RNAse H is not required, under certain conditions, the RNAse Hactivity present in AMV reverse transcriptase may be inhibited bycomponents present in the reaction mixture. In such situations, additionof exogenous RNAse H may be desirable. Where relatively large amounts ofheterologous DNA are present in the reaction mixture, the native RNAse Hactivity of the AMV reverse transcriptase may be somewhat inhibited (seee.g., Example 8) and thus the number of copies of the target sequenceproduced accordingly reduced. In situations where the target sequencecomprises only a small portion of DNA present (e.g., where the samplecontains significant amounts of heterologous DNA), it is particularlypreferred to add exogenous RNAse H. One such preferred RNAse H is E.coli RNAse H. Addition of such exogenous RNAse H has been shown toovercome inhibition caused by large amounts of DNA. (See, e.g., Example8).

The RNA transcripts produced by these methods may serve as templates toproduce additional copies of the target sequence through theabove-described mechanisms. The system is autocatalytic andamplification by the methods of the present invention occursautocatalytically without the need for repeatedly modifying or changingreaction conditions such as temperature, pH, ionic strength or the like.This method does not require an expensive thermal cycling apparatus, nordoes it require several additions of enzymes or other reagents duringthe course of an amplification reaction.

The methods of the present invention may be used as a component ofassays to detect and/or quantitate specific nucleic acid targetsequences in clinical, environmental, forensic, and similar samples orto produce large numbers of copies of DNA and/or RNA of specific targetsequence for a variety of uses.

In a typical assay, a sample to be amplified is mixed with a bufferconcentrate containing the buffer, salts, magnesium, triphosphates,primers and/or splice templates, dithiothreitol, and spermidine. Thereaction may optionally be incubated near 100° C. for two minutes todenature any secondary structures in the nucleic acid. After cooling, ifthe target is a DNA target without a defined 3′ terminus, reversetranscriptase is added and the reaction mixture is incubated for 12minutes at about 42° C. The reaction is again denatured near 100° C.,this time to separate the primer extension product from the DNAtemplate. After cooling, reverse transcriptase, RNA polymerase, andRNAse H are added and the reaction is incubated for two to four hours at37° C. The reaction can then be assayed by denaturing the product,adding a probe solution, incubating 20 minutes at 60° C., adding asolution to selectively hydrolyze the label of the unhybridized probe,incubating the reaction six minutes at 60° C., and measuring theremaining chemiluminescent label in a luminometer. The methods of theHPA application cited supra are incorporated herein. Several othermethods for product determination may be employed in place of thein-solution probe hybridization.

If the target has a defined 3′ terminus and one of the oligonucleotidesis a splice template which has a complexing sequence sufficientlycomplementary to the 3′ terminus to complex therewith and a promotersequence 5′ to the complex sequence or the target is RNA, a typicalassay includes mixing the target with the buffer concentrate mentionedabove and denaturing any secondary structure. After cooling, reversetranscriptase, RNA polymerase, and if desired, RNAse H are added and themixture is incubated for two to four hours at 37° C. The reaction canthen be assayed as described above.

II. Preliminary Procedures

The following are several embodiments of preliminary procedures whichoptionally may be employed in conjunction with the preferred method ofthe present invention. Since some target nucleic acids requiremodification prior to autocatalytic amplification, these procedures maybe employed to accomplish the modifications. Where the target nucleicacid (and target sequence) is originally DNA, these procedures may beemployed to produce RNA copies of the target sequence for use in theGeneral Method. It should be appreciated that these PreliminaryProcedures may themselves be repeated and therefore may be used asamplification methods in their own right.

Preliminary Procedure I

This method gives RNA copies of a target sequence of a target nucleicacid which comprises a (single-stranded) DNA with a defined 3′ terminus.Preliminary Procedure I uses two nucleic acid components: a targetnucleic acid molecule and an oligonucleotide splice template. Thisprocedure requires a DNA target nucleic acid having a defined 3′-end. Ifthe native 3′ terminus is not known or is unsatisfactory for any reason,a new defined 3′ terminus may be created by use of a restrictionnuclease, ligation to another sequence, or some other means.

In the following description, (see FIGS. 2A to 2C) the target nucleicacid will arbitrarily have the “minus” sense. Thus, the splice templatewill have the “plus” sense so as to be sufficiently complementary to thetarget to complex therewith. The splice template has a complexingsequence sufficiently complementary to the 3′ terminus of the target tocomplex therewith. The splice template also has a sequence 5′ to thecomplexing sequence which includes a promoter sequence for an RNApolymerase. The splice template may optionally have other sequences 5′to the promoter, between the promoter and complexing sequences, and/or3′ to the complexing sequence. The splice template may also be modifiedat the 3′ terminus to be “blocked” so that it cannot be extended byadding additional nucleotides in an extension reaction and is renderedincapable of acting as a primer in addition to acting as a splicetemplate.

Preliminary Procedure I uses two enzyme activities: a DNA-dependent DNApolymerase and a DNA-dependent RNA polymerase.

The target nucleic acid is treated with the splice template underconditions wherein an oligonucleotide/target sequence complex is formedand DNA synthesis may be initiated. In a DNA extension reaction using anappropriate DNA polymerase, a sequence complementary to the sequence ofthe splice template 5′ to the complexing sequence is added to the 3′terminus of the target DNA. The splice template, if not blocked at the3′ terminus, may also serve as a primer for the DNA polymerase and beextended to give a primer extension product. The product of theextension reaction, either double-stranded or partially double-stranded,target/splice template complex acts as a template for the synthesis RNAtranscripts using an RNA polymerase which recognizes the promoter. TheRNA transcripts may be then used for the general method or be used togenerate DNA copies of the RNA as follows:

An RNA transcript comprising the target sequence (having the “plus”sense) is treated with a primer (which nominally has the “minus” sense)which has a complexing sequence sufficiently complementary to the 3′ endof the target sequence of the RNA transcript to complex therewith underconditions whereby an oligonucleotide/target sequence complex is formedand DNA synthesis may be initiated. The primer is then extended in a DNAextension reaction using the RNA transcript as template to generate aDNA primer extension product having the target sequence. The DNA targetsequence is separated from the RNA transcript by either denaturation orby degradation of the RNA and beginning with the splice template, thecycle is repeated. Optionally, the primer may also have additionalsequences 5′ to the priming sequence. The splice template may also haveadditional sequences 3′ to the complexing sequence.

In one embodiment, the above method may be practiced using oneoligonucleotide by using an oligonucleotide having a sequence whichwould comprise the primer 3′ to the sequence which would comprise thesplice template. (See, e.g. FIG. 2C).

Preliminary Procedure I is further described by reference to FIGS. 2A to2E. FIG. 2A depicts a target nucleic acid (DNA) which has a defined 3′terminus, having no additional sequences 3′ to the target sequence. Thefirst oligonucleotide comprises both a primer and a splice template andhas a promoter 5′ to its complexing sequence.

FIG. 2B depicts a target nucleic acid (DNA) as shown in FIG. 2A. Thefirst oligonucleotide comprises a splice template which is blocked atits 3′ end and is thus incapable of acting as a primer.

FIG. 2C depicts a target DNA as shown in FIGS. 2A and 2B. FIG. 2Cdepicts the use of one oligonucleotide which has a splice template (witha promoter) sequence 5′ to a primer sequence at its 3′ end. Thus, theoligonucleotide acts as a blocked splice template in steps (1) and (7)and as a primer (and splice template) in step (4).

FIG. 2D depicts a target nucleic acid (DNA) which has a defined 5′-endand additional sequences 3′ to the target sequence which undergoesprefatory complexing, primer extending and separating steps (steps 1 and2) to generate a complementary DNA target having a defined 3′-terminus.The oligonucleotide of steps (1) and (7) comprises a primer whichcomplexes with the 3′ terminal portion of the target sequence of theoriginal target DNA (here nominally (+)). The other oligonucleotide (ofsteps (4) and (10)) comprises an unblocked splice template whichcomplexes with the 3′-end of the complement of the original target.

FIG. 2E depicts a target nucleic acid (DNA) which has a defined 5′terminus and additional sequences 3′ to the target sequence whichundergoes prefatory complexing, extending and separating steps (steps(1) and (2)) to generate a complementary DNA target having a defined3′-terminus. The oligonucleotide of steps (1) and (7) comprises a primerwhich complexes with the 3′ terminal portion of the target sequence ofthe original target DNA (here nominally (+)). The oligonucleotide ofsteps (4) and (10) comprises a blocked splice template which complexeswith thed 3′ terminal portion of the target sequence of the complementof the original target.

The splice template is complexed with the 3′ terminus of the targetnucleic acid under complexing conditions so that a target/splicetemplate complex is formed and DNA synthesis may be initiated. (step 1)A suitable DNA polymerase is used to extend the 3′ terminus of thetarget nucleic acid to add a sequence complementary to the sequence ofthe splice template 5′ to the complexing sequence. If the 3′ terminus ofthe splice template has not been blocked, and is sufficientlycomplementary to the target nucleic acid the splice template may act asa primer so that the 3′ terminus of the splice template may also beextended. (FIG. 2A) The resulting target/splice template complex may beeither partially or completely double-stranded. (See FIG. 2A versusFIGS. 2B and 2C). At minimum the double-stranded region comprises thepromoter sequence and the priming sequence which complexed with thetarget nucleic acid.

The template of Step 2 is transcribed by an appropriate RNA polymerase.The RNA polymerase produces about 5-1000 copies of RNA for eachtemplate. In FIGS. 2A to 2C, the RNA produced is nominally of the “plus”sense and includes the sequence from the 3′ end of the promoter to the5′ end of the target nucleic acid.

The RNA product of Step 3 may be used according to the general method toautocatalytically amplify the target sequence or alternatively may betreated under complexing and priming conditions with a primer which hasa complexing sequence at its 3′ terminus sufficiently complementary tothe 3′ terminal portion of the target sequence in the RNA to complextherewith and optionally includes other sequences 5′ to the complexingsequence. The primer is extended using the RNA as a template by anRNA-dependent DNA polymerase in a primer extension reaction. Thisproduces a product which is at least partially double-stranded and whichmust be made at least partially single-stranded for further synthesis bydegradation of the RNA portion, such as by an RNAse H (step 5) or bysome other method such as denaturation.

The DNA produced in step 6 may be used as a target molecule for Step 1and treated with a splice template as described above to produce moreRNA and DNA. These steps may be cycled to produce any desired level ofamplification. It should also be noted that, by appropriate choice ofthe splice template and primer(s), this new target molecule (Step 6) mayhave different termini than the original molecule. Furthermore, if theprimer extension product from the RNA has a promoter sequence 5′ to thecomplexing sequence, a second primer having a complexing sequence of the“plus” sense and optionally other sequences 5′ to the complexingsequence may be used to copy the extended primer product to produce adouble-stranded template for an RNA polymerase. A template so producedenables the RNA polymerase to make “minus” sense RNA which may beamplified using the general method or further amplified using theprocedures herein.

Preliminary Procedure II

Preliminary Procedure II differs from Preliminary Procedure I in the waythe autocatalytic species is generated. The DNA target nucleic acid neednot have a defined 3′ terminus. In one aspect, a primer containing apromoter sequence 5′ to the complexing sequence is used instead of asplice template to introduce the promoter sequence into the template forthe RNA polymerase. The primer has a complexing sequence sufficientlycomplementary to the 3′-terminal portion of the target sequence tocomplex therewith and a sequence which includes a promoter for an RNApolymerase 5′ to the complexing sequence. The primer is extended with aDNA polymerase to give a primer extension product.

After separation of the strands, usually by thermal denaturation, asecond oligonucleotide of the same sense as the target molecule is usedas a primer to synthesize a DNA complement to the first primer extensionproduct. The second primer may have additional sequences 5′ to thecomplexing region which may include a promoter. Inclusion of a promotersequence in the second primer may enhance amplification. The secondprimer may also be a splice template.

Preliminary Procedure II is further described with reference to FIG. 3.FIG. 3 depicts a target DNA which has additional sequences both 5′ and3′ to the target sequence. The first primer has a complexing sequence atits 3′ terminus and a sequence 5′ to the complexing sequence whichincludes a promoter sequence and complexes sufficiently with the targetto serve a priming function and initiate DNA synthesis. An appropriateDNA polymerase extends the primer to give a primer extension product.The strands are separated by denaturation or other means to yield to asingle-stranded promoter containing primer extension product.

A second primer is used which has a complexing sequence at its 3′terminus sufficiently complementary to the 3′ terminal portion of thetarget sequence of the first primer extension product and, optionally,other sequences 5′ to the complexing sequence which may include apromoter sequence. The second primer is complexed sufficiently with theprimer extension product from Step 3 to serve a priming function andinitiate DNA synthesis. The DNA polymerase extends the second primer togive a second primer extension product. The resulting double-strandedDNA molecule may now serve as a template for the RNA polymerase togenerate RNA transcripts for the General Method. As depicted in FIG. 3,the RNA molecules produced are nominally of the “plus” sense and may bemultiplied using the general method of the present invention.

Where the complexing sequence of the second primer is complementary tothe 3′ terminus of the first primer extension product from Step 3 andthe second primer includes a promoter sequence 5′ to the complexingsequence, the second primer may serve as a splice template so that the3′-terminus of the first primer extension product from Step 3 may befurther extended to add the promoter sequence and produce a template forthe RNA polymerase which produces RNA transcripts of both senses. TheRNA molecules so produced may be amplified using the general method.

In another aspect of the present invention, the second primer acts as asplice template and has a promoter 5′ to the complexing sequence, sothat the first primer need not have a promoter. In that case, the firstprimer extension product from step 2 is further extended to produce acomplement to the promoter sequence, thus generating a template for theproduction of “minus” sense RNA by the RNA polymerase.

By repeating the steps described above, additional RNA and DNA copies ofthe target sequence may be produced,

EXAMPLES

Preface

The following examples of the procedures previously describeddemonstrate the mechanism and utility of the methods of the presentinvention. They are not limiting to the inventions and should not beconsidered as such.

Many of the materials used in one or more examples are similar. Tosimplify the descriptions in the examples, some of the materials will beabbreviated and described here.

The template referred to as “frag 1” is a double-stranded segment of DNAhomologous to a region of DNA from the hepatitis B genome. It has beenexcised from a plasmid via restriction nuclease digestion and purifiedfrom the remaining nucleic acid by chromatographic methods. Subsequentto purification, the fragment has been cut with a restrictionendonuclease and purified via phenol extraction and ethanolprecipitation to yield the desired target.

The template referred to as “M13L(−)” is a purified single-stranded DNAtarget containing, as a small fraction of the total sequence, the minusstrand target sequence.

Several different primers and splice templates were used in the examplesdescribed herein. The oligonucleotides referred to as T7pro(+) contains,near the 5′ terminus, a T7 RNA polymerase promoter and, near the 3′terminus, a sequence complementary to the 3′ terminus of theminus-strand target sequence to the two templates described above;T7pro(+) also contains other sequence information to enhanceperformance. The sequence for T7pro(+) is 5′-AATTT AATAC GACTC ACTATAGGGA GAGGT TATCG CTGGA TGTGT CTGCG GCGT3′ (SEQ ID NO:26).

Another oligonucleotide similar to T7pro(+) is ddT7pro(+). It differsfrom T7pro(+) in that the 3′ terminus has been extended with a dideoxynucleotide using terminal deoxynucleotidyl transferase. Unlike T7pro(+),ddT7pro(+) is incapable of serving as a primer for DNA synthesis byreverse transcriptase but can act as a splice template.

HBV(−)Pr is a primer which will hybridize to the plus strand of the frag1 template and is homologous to a sequence within the M13L(−). [HBV(−)Pris complementary to a sequence 3′ to the plus strand sequence homologousto the T7pro(+).] The sequence for HBV(−)Pr is 5′-GAGGA CAAAC GGGCAACATA CCTTG-3′ (SEQ ID NO:27).

Another oligonucleotide containing a promoter region is T7pro(−). Thispromoter-primer contains a sequence identical to T7pro(+) but replacesthe sequence complementary to the minus target with a sequencecomplementary to the plus target. The 3′ terminus of T7pro(−) iscomplementary to the 3′ terminus of the plus strand of frag 1. Thesequence for T7pro(−) is 5′-AATTT AATAC GACTC ACTAT AGGGA GATCC TGGAATTAGA GGACA AACGG GC-3′ (SEQ ID NO:28). Like the ddT7pro(+), ddT7pro(−)is a 3′ blocked oligonucleotide made by extending the 3′ terminus with adideoxynucleotide using terminal deoxynucleotidyl transferase. TheddT7pro(−) cannot serve as a primer but is otherwise similar toT7pro(−).

The templates used in these examples contain substantial sequencebetween the regions homologous or complementary to the and splicetemplates described above. As the sequence between the oligonucleotideswill be amplified as a result of the invention, quantification of thissequence provides a specific means of measuring the amplification. Ithas been convenient to assay the products by hybridization techniquesusing DNA probes specific for the sequences coded for between theoligonucleotide primers and splice templates. Two probes are used in theexamples presented below: Probe(+) and Probe(−). Probe(+) iscomplementary to the minus sense product and Probe(−) is complementaryto the plus sense product. The sequence for Probe(+) is 5′-CCTCT TCATCCTGCT GCTAT GCCTC-3′ (SEQ ID NO:29) and the sequence for Probe(−) is5′-GAGC ATAGC AGCAG GATGA AGAGG-3′ (SEQ ID NO:30). The probes usedherein have been labeled with a chemiluminescent tag. In the assay, thelabel on hybridized probe emits light which is measured in aluminometer.

In the following examples, relative amplification was measured asfollows. A sample of the amplification reaction mixture (usually 10 μl)was diluted to 50 μl with 10 mM Tris-HCl, pH 8.3, and denatured twominutes at 95° C. After cooling on ice, 50 μl of a probe solutioncontaining approximately 75 fmol Probe(+) or Probe(−), 0.2 M lithiumsuccinate, pH 5.2, 21% (w/v) lithium lauryl sulfate, 2 mM EDTA, and 2 mMEGTA, was added to the sample and mixed. The reactions were thenincubated 20 minutes at 60° C. and cooled. To each hybridizationreaction was added 500 μl of a solution prepared by adjusting asaturated sodium borate solution to pH 8.5 with hydrochloric acid, thendiluting to bring the borate concentration to 0.8 M final and addingTriton X-100 to 5% (v/v) final. The reactions were then mixed andincubated six minutes at 60° C. to destroy the chemiluminescent label ofthe unhybridized probe. This method of destruction of thechemiluminescent label of unhybridized probe is quite specific; only avery small fraction of the unhybridized probe remains chemiluminescent.The reactions were cooled and the remaining chemiluminescence wasquantified in a luminometer upon the addition of 200 μl of 1.5 M sodiumhydroxide, 0.1% (v/v) hydrogen peroxide. In the assay, hybridized probeemits light which is measured in a luminometer. Since the reaction whichdestroys the chemiluminescent label of unhybridized probe is not 100%effective, there is generally a background level of signal present inthe range of about 300 to 1300 relative light units (RLU).

Many other assay methods are also applicable, including assays employinghybridization to isotopically labeled probes, blotting techniques andelectrophoresis.

The enzymes used in the following examples are avian myeloblastosisvirus reverse transcriptase from Seikagaku America, Inc., T7 RNApolymerase from New England Biolabs or Epicentre, and Moloney murineleukemia virus (MMLV) reverse transcriptase and E. coli RNAse H fromBethesda Research Laboratories. Other enzymes containing similaractivities and enzymes from other sources may be used; and other RNApolymerases with different promoter specificities may also be suitablefor use.

Unless otherwise specified the reaction conditions used in the followingexamples were 40 mM Tris-HCl, 25 mM NaCl, 8 mM MgCl₂, 5 mMdithiothreitol, 2 mM spermidine trihydrochloride, 1 mM rATP, 1 mM rCTP,1 mM rGTP, 1 mM rUTP, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mMdTTP, 0.15 μM each primer or splice template, and specified amounts oftemplate and enzymes in 100 μl volumes.

These reaction conditions are not necessarily optimized, and have beenchanged as noted for some systems. The oligonucleotide sequences usedare exemplary and are not meant to be limiting as other sequences havebeen employed for these and other target sequences.

Example 1 Preliminary Procedure I

To demonstrate that this system worked, each of the four promotercontaining oligonucleotides described above were each respectively putinto reactions with or without 4 fmol target (frag 1). The reaction wasincubated 2 minutes at 95° C. to denature the target, then cooled toallow the oligonucleotides to anneal to the target. Reversetranscriptase, 13 Units, and T7 RNA polymerase, 100 Units, were addedand the reaction was incubated 30 minutes at 37° C. One-tenth of thereaction was assayed using the hybridization method. The results (inRelative Light Units or “RLU(s)” and fmols) presented in Table 1 showthat both the blocked and unblocked oligonucleotides serve as splicetemplates to produce product. The signals measured for reactions withouttarget represent typical background levels of signal, for this type ofassay.

TABLE 1 Comparison of Splice Templates in Preliminary Procedure I. SpiceProduct Template Target Probe(−) RLU fmol ddT7pro(+) 4 fmol Probe(−)38451 240 ddT7pro(+) 0 fmol Probe(−) 544 0 T7pro(+) 4 fmol Probe(−)65111 410 T7pro(+) 0 fmol Probe(−) 517 0 ddT7pro(−) 4 fmol Probe(+)47756 85 ddT7pro(−) 0 fmol Probe(+) 879 0 T7pro(−) 4 fmol Probe(+)156794 290 T7pro(−) 0 fmol Probe(+) 600 0

Example 2 Cycling with Preliminary Procedure I

The amplification system was cycled with ddT7pro(+) and T7pro(+). Inthis experiment, 4 amol frag I, HBV(−)Pr and ddT7pro(+) or T7pro(+) weremixed in standard reactions and incubated at 95° C. After cooling, 13Units of reverse transcriptase and 100 units of T7 RNA polymerase wereadded and the mixture was incubated 30 minutes at 37° C. One-tenth ofthe reaction was removed for assay and the cycle was repeated with theremainder. After repeating the cycle a third time, the 10 μl aliquotswere assayed by the hybridization method using Probe(−). The resultspresented in Table 2 indicate that product is amplified through cyclingwith both blocked and unblocked splice templates.

TABLE 2 Cycling with Preliminary Procedure I. Relative LightUnits(RLU's) Splice template Target Cycle 1 Cycle 2 Cycle 3 ddT7pro(+) 4amol 602 1986 10150 ddT7pro(+) 0 amol 658 592 595 T7pro(+) 4 amol 8916180 52160 T7pro(+) 0 amol 496 504 776

Example 3 Sensitivity of Preliminary Procedure I

In this example the unblocked splice template, T7pro(+), and the primer,HBV(−)Pr, were used to test the sensitivity of the amplification method.Six cycles of Preliminary Procedure I were run as described in Example 2with decreasing quantities of frag 1. After amplification, the productwas assayed using the hybridization method described in the DetailedDescription of the Invention. Using this method, 4×10⁻²¹ moles frag 1could be detected (see Table 3).

TABLE 3 Sensitivity using 6 cycles of Preliminary Procedure I. TargetSample Product (moles) μl RLU's 4 × 10⁻¹⁸ 5 328390 4 × 10⁻¹⁹ 20 105364 4× 10⁻²⁰ 20 3166 4 × 10⁻²¹ 20 1927 0 20 806

Example 4 Amplification Including Preliminary Procedure I

In the following example, the target to be amplified was frag 1. In thefirst set of reactions, various combinations of target and splicetemplates were incubated at 95° C. for two minutes then cooled prior toadding 13 Units of reverse transcriptase and 100 Units of T7 RNApolymerase. The reactions were incubated 30 minutes at 37° C. then 5 μlaliquots of the reactions were assayed with both probes to quantitatethe products. Subsequent to this assay, reactions were prepared using 5μl of reactions 1 and 2 and the T7pro(−) splice template. The mixtureswere incubated 2 minutes at 95° C. then cooled prior to adding 13 Unitsof reverse and 100 Units of T7 RNA polymerase. The new reactions werethen mixed and incubated at 37° C. for 2 hours. Aliquots of 10 μl wereremoved to an ice bath at time points indicated in Table 4 below. Theproducts were assayed using the hybridization method previouslydescribed. The data indicate that both splice templates allow productionof RNA from frag 1. The data also indicate significantly more minus andplus sense products are produced in the reactions containing RNA and thesplice template complementary to that RNA. And, finally, the reactionkinetics for the reaction 1B show a geometric increase in productwhereas the kinetics for the 2B reaction are of a more linear form. Thisdifference indicates the product in the 1B reaction is serving as asubstrate to generate more product; thus the reaction is autocatalytic.

TABLE 4 Preliminary Procedure I Reaction 1 2 3 4 Target (4 fmol) Yes YesYes No T7pro(+) Yes No No No T7pro(−) No No Yes No Probe Time RelativeLight Units(RLU's) Probe(+) 30′ 1156 1058 21859 591 Probe(−) 30′ 11693771 744 691 Reaction 1A 1B 2A 2B Reaction1 Yes Yes No No Reaction2 No NoYes Yes T7pro(−) No Yes No Yes Probe Time Relative Light Units Probe(+) 0′ 714 757 639 661  30′ 686 339 1663 1373  60′ 718 6331 645 1786 120′816 16889 660 2238 Probe(−) 120′ 3142 6886 637 780

Example 5 Reaction Kinetics for Amplification Including PreliminaryProcedure I

The following example further demonstrates the potential of thisembodiment of the methods the invention. A small quantity of frag 1 (10amol), was used in reactions with various combinations of T7pro(+),T7pro(−), and HBV(−)Pr. The reaction mixtures were incubated at 95° C.for two minutes to denature the DNA target and cooled prior to addingreverse transcriptase and T7 RNA polymerase. After mixing, the reactionswere incubated 30 minutes at 37° C. Fifteen microliter aliquots wereremoved at various time points and stored at 0° C. until assayed. Thehybridization assay was used to quantitate the products of thereactions. The data presented in Table 5 show that the inventionrequires one splice template and one primer. A second splice template isadvantageous, however. The results with only one primer or splicetemplate were below the detection limits of the assay. As in theprevious example, the reaction kinetics are geometric, indicating anautocatalytic system.

TABLE 5 Preliminary Procedure II Reaction Kinetics. Reaction 1 2 3 4 5 67 Target (10 amol) No Yes Yes Yes Yes Yes Yes T7pro(+) Yes No Yes No NoYes Yes T7pro(−) Yes No No Yes No No Yes HBV(−)Pr Yes No No No Yes YesNo Time (minutes) Minus Product (RLU's) 0 619 638 635 703 592 619 656 30613 635 613 755 626 844 1133 60 635 649 856 894 635 2146 6008 90 593 619619 925 624 6226 23484 120 621 606 627 946 639 12573 43939 180 678 635714 930 627 21719 78682 Time (minutes) Plus Product (RLU's) 0 624 6461661 710 621 636 962 30 637 601 602 629 655 803 758 60 639 706 800 679664 226 2895 90 638 683 956 633 687 7786 8085 120 643 670 884 647 63218160 18241 180 683 617 968 758 712 34412 41165

Our subsequent work has demonstrated continued product synthesis forover 5.0 hours, which is substantially better than prior art methods. Wealso have demonstrated increased sensitivity.

Example 6 Amplification Including Preliminary Procedure II

In this example various combinations of primers were used to amplify 500amol of a DNA target without defined termini. The target was the M13L(−)referenced above. Upon reaction preparation, the samples were incubatedtwo minutes at 95° C., then cooled prior to adding 13 Units of reversetranscriptase. The reactions were then incubated twelve minutes at 42°C. Next the reactions were again heated for two minutes at 95° C. andcooled. Reverse transcriptase and T7 RNA polymerase were added and thereactions were incubated for two hours at 37° C. Ten microliter aliquotsof the reaction were assayed with both Probe(+) and Probe(−). Theresults presented in Table 6 show that synthesis of large amounts ofnucleic acid occurs only when two primers are employed. They alsodemonstrate the benefit of two promoter-primers over one promoter-primerand one primer. The low level synthesis in reactions 4 and 5 correspondto synthesis of approximately one copy of DNA from the originaltemplate. The system employs an initial 95° C. denaturation step whichmay serve to denature double-stranded targets or double-stranded regionsof a single-stranded target as well as inactivate unwanted nuclease andprotease activities.

TABLE 6 Preliminary Procedure II System. Reaction 1 2 3 4 5 6 7 M13L(−)No No Yes Yes Yes Yes Yes T7pro(+) Yes Yes No Yes No Yes Yes T7pro(−)Yes No No No Yes Yes No HBV(−)Pr No Yes No No No No Yes Probe RelativeLight Units (RLU's) Probe(+) 862 744 762 1089 2577 96221 30501 Probe(−)473 420 483 3038 1080 15171 14863

Example 7 Effect of RNAse H

To demonstrate that the addition of exogenous RNAse H may improveamplification in the autocatalytic systems described of the presentinvention, several reactions were prepared using various quantities oftarget, M13L(−), and either 0 or 2 Units of exogenous RNAse H. Theexogenous RNAse H used was derived from E. coli. All reactions wereprepared with T7pro(+) and T7pro(−). The reactions were subjected to the95° C. denaturation and cooled prior to adding reverse transcriptase.After a twelve minute incubation at 42° C., the reactions were againdenatured at 95° C. After cooling, reverse transcriptase, T7 RNApolymerase, and, if indicated (see Table 7), RNAse H was added and thereactions were incubated for 3 hours at 37° C. Aliquots of 10 μl wereremoved from each reaction at hourly intervals for assay by thehybridization method. The data in Table 7 show that exogenous RNAse Hsignificantly enhanced the reaction kinetics and increases thesensitivity of the invention. Signals less than 600 RLUs wereinterpreted as typical background levels.

TABLE 7 Effect of Exogenous RNAse H on Amplification IncludingPreliminary Procedure II Sensitivity. Target RNAse H RLU's atTime(hours) (moles) (Units) 0 1 2 3 1 × 10⁻¹⁷ 0 478 7659 28716 60443 1 ×10⁻¹⁸ 0 440 946 13332 36039 1 × 10⁻¹⁹ 0 413 581 10063 41415 1 × 10⁻²⁰ 0406 424 717 4520 0 0 455 376 579 2075 1 × 10⁻¹⁸ 2 419 20711 50389 640731 × 10⁻¹⁹ 2 411 6831 21312 29818 1 × 10⁻²⁰ 2 420 604 1281 1375

Example 8 Effect of Exogenous DNA

It has been demonstrated that the addition of exogenous DNA maysignificantly inhibit this autocatalytic amplification system. Tofurther demonstrate the benefit of adding exogenous RNAse H,amplification reactions were prepared with or without 2 μg calf thymusDNA to demonstrate this inhibition. In reactions with the calf thymusDNA, two concentrations of reverse transcriptase were employed to testwhether additional AMV RNAse H would overcome the inhibition. Also,RNAse H from E. coli was added to some of the reactions for the samereasons. The reactions differ from the standard reactions in that theconcentration for each of the ribonucleotides were increased to 2.5 mMand the concentration of magnesium chloride was increased to 12.8 mM.The reactions were prepared using 100 amol of M13L(−) as a target andT7pro(+) and T7pro(−). After denaturing two minutes at 95° C., thereactions were cooled and 13 or 39 Units of reverse transcriptase wereadded and the reactions were incubated 12 minutes at 37° C. Thereactions were again denatured at 95° C. and cooled prior to adding 13or 39 Units of reverse transcriptase, 100 or 300 units of T7 RNApolymerase, and either 0 or 2 Units of E. coli RNAse H. After incubatingone hour at 37° C., 10 μl of each reaction was assayed using thehybridization assay. The results presented in Table 8 showed that thecalf thymus DNA inhibited the reaction by 90% in comparison to areaction system without exogenous DNA and that additional reversetranscriptase (and its associated RNAse H) did not significantly affectthe product amplification. The addition of more T7 RNA polymerase didhave a significant effect on the product yield, but it was smallrelative to the increase due to addition of exogenous RNAse H. Not onlywas the inhibition eliminated, the amplification was increased overfive-fold relative to the reaction without the calf thymus DNA and E.coli RNAse H. The signals observed with the higher amount of E. coliRNAse H were saturating for the amount of probe used in thehybridization assay. To accurately quantitate these samples, dilution ofthe amplified product would be required before assaying.

TABLE 8 Effect of RNAse H on Amplification Inhibition by Exogenous DNAReverse T7 RNA E. coli Target Exogenous Relative TranscriptasePolymerase RNAse H DNA DNA Light (Units) (Units) (Units) (amol) (μg)Units 13 100 0 0 0 458 13 100 0 100 0 55305 13 100 0 100 2 3003 39 100 0100 2 2786 13 300 0 100 2 5434 39 300 0 100 2 6831 13 100 4 100 2 27866639 100 4 100 2 334649 13 300 4 100 2 359101 39 300 4 100 2 375043

Example 9 Amplification by the General Method

This system does not require an initial transcription and denaturation;a DNA complement of the target is made and the original target isremoved by the RNAse H. The DNA may then anneal to a second primer orpromoter-primer and through DNA synthesis produce a template for the RNApolymerase. If the RNAse H is not active, the DNA:RNA hybrid producedfirst will end the reaction without producing a template for the RNApolymerase. In an attempt to demonstrate the method of this invention, aplasmid containing an RNA polymerase promoter and the target nucleicacid was used to produce large quantities of single-stranded RNAtranscripts containing the target sequence within other sequences. Twosimilar reactions were also prepared: one containing the plasmid withoutthe RNA polymerase and the other with the RNA polymerase without theplasmid. Dilutions of each of these reactions were assayed to quantitatethe products. Equivalent dilutions of all three reactions were used toprepare amplification reactions containing the two promoter-primers, T7pro(+) and T7 pro(−). Reaction 1 of Table 9 contained 60 amol of RNA and0.6 amol of the starting plasmid. Reaction 2 contained 0.6 amol of thestarting plasmid, but no RNA. Reaction 3 contained no target. Thereactions were not denatured at 95° C.; instead reverse transcriptaseand T7 RNA polymerase were added and the reaction was incubated at 37°C. for four hours. Aliquots were removed hourly for later assay by thehybridization method. As shown in Table 9, the reaction containing theplasmid and the RNA produced from the plasmid gave large hybridizationsignals; the reaction containing the plasmid alone produced significantproduct as the T7 RNA polymerase could produce RNA from the plasmidwhich could then be utilized according to the General Method to producemore nucleic acid of both senses; and, finally, the control (Reaction 3)containing no target produced nothing.

TABLE 9 Preliminary Procedure IV Reaction Kinetics. Time Reaction 1 2 31 2 3 (hours) RLU's for Probe(+) RLU's for Probe(−) 2 9695 1890 886 79791074 434 3 22266 2819 824 15018 1487 491 4 33863 4571 628 16540 2310 556Rxn 1: plasmid plus RNA. Rxn 2: plasmid. Rxn 3: no target

Example 10

Amplification by the General Method

The following experiment was done to determine if other methods ofinitiation were possible for the invention which may alleviate the needfor the first primer extension and denaturation step for DNA targetswithout defined termini. In this experiment, the stated quantities oftarget are amplified with or without the first reverse transcriptasestep as indicated in Table 10. The amplification time once all enzymesare added was four hours. The target used was the M13(+) diluted intonormal saline. T7pro(+) and T7pro(−) were used. To 25 μl of eachdilution was added 25 μl 0.1N KOH. The samples were incubated tenminutes at 98° C. and then cooled. Each sample was brought to 100 μl andfinal concentrations of 50 mM Trizma base, 40 mM glutamic acid, 25 mMpotassium hydroxide, 12.8 mM magnesium chloride, 5 mM dithiothreitol, 2mM spermidine trihydochloride, 2.5 mM each ribonucleotide triphosphate,0.2 mM each deoxyribonucleotide triphosphate, and 0.15 μM each primer.To the Standard Protocol tubes, 13 U reverse transcriptase was added andthe reactions were incubated 12 minutes at 42° C., then two minutes at95° C., and cooled. Then to all tubes was added 13 U reversetranscriptase, 100 U T7 RNA polymerase, and 0.5 U RNAse H. The reactionswere then incubated four hours at 37° C. and 10 μl aliquots were assayedusing the chemiluminescent probe assay. The results presented in Table10 show that some amplification was evident using the abbreviatedprotocol. And although the level of amplification observed wassignificantly less than that for the Standard Protocol, this may befurther developed to be as efficient or may be useful in cases wheresignificant levels of target nucleic acid are present.

TABLE 10 Amplification Without First Primer Extension Target MolesProtocol RLU's M13(+) 3.8E-17 Standard 2295451 ″ 3.8E-19 ″ 2374443 ″3.8E-21 ″ 230227 Negative 0 ″ 3037 M13(+) 3.8E-17 Short 2475574 ″3.8E-19 ″ 27209 ″ 3.8E-21 ″ 17144 Negative 0 ″ 1679

The likely explanation for these data is that T7RNA polymerase is notcompletely specific. There is a low level of random RNA synthesis whichgenerates small numbers of RNA copies of the target regions. Thesecopies are amplified via the standard method.

Our initial work demonstrated excellent amplification with certainprimer sets and targets without the addition of exogenous RNAse H. Oursubsequent work clarifying the mechanism of the reaction has made itpossible to efficiently apply the method to a wider variety of targets.We disclose and claim herein methods which both use exogenous RNAse H inamplification and those which rely on RNAse H activity associated withreverse transcriptase.

We have discovered that E. coli RNAse should not routinely be added asit does not always improve amplification. We have determined that someforms of RNAse H are sequence specific as to where they cut the RNA ofRNA:DNA hybrids. In the amplification reaction, we have not detected thepromoter-containing primer in full-length product DNA. In embodimentsusing two promoter-primers, only one of the promoter primers isdetectably incorporated into full-length product DNA. All othermechanisms that have been postulated by those skilled in the art showfull-length product DNA containing both primers. We attribute thesefindings to the likelihood that RNAse H is not fully degrading the majorRNA species synthesized during amplification. Based on these findings, anew amplification mechanism is set forth herein which incorporates ourfindings regarding RNAse H sequence specificity. New and usefulpromoter-primer design criteria are disclosed. Furthermore, we claimherein novel methods for synthesizing multiple copies of a targetnucleic acid sequence, comprising

1) selecting a primer, complementary to a portion of the RNA targetsequence, which complexes with the portion of the RNA target, saidportion of the target located such that it remains capable of formingprimer extension product after being exposed to degradation by aselected RNAse H;

2) selecting a promoter-primer complementary to a portion of the DNA tobe obtained by extension of the primer, which complexes with the DNA inan area where substantially all of the complementary RNA is removed fromthe duplex through degradation of RNAse H; and

3) combining the RNA target with the primer, promoter-primer, RNAse H,reverse transcriptase and transcriptase and forming multiple copies ofthe RNA and multiple copies of DNA complementary to the RNA. The novelmethods herein described do not make a substantially equivalent numberof copies of DNA of the same polarity (or “sense”) as the RNA targetsequence.

This procedure provides a method which permits the design of efficientpromoter-primers and primers for new target sites. We disclose and claimherein such promoter-primer and primer combinations and design criteriafor same. The mechanism disclosed herein involves a novel reactionintermediate for transcription of RNA strands. This intermediate is adouble-stranded complex of RNA and DNA which contains a double-strandedDNA promoter region. Nothing like this has, to our knowledge, ever beendescribed in the literature. Indeed, none of the prior art systems,specifically neither Guatelli, J. C. et al., 87 PNAS 1874-1878 (1990),nor PCT App. Ser. No. 88/10315 to Gingeras, T. R. et al., properlyselect the primer and promoter-primer sequence based upon the locationof RNAse H degradation sites. Thus, the methods herein are novel andnonobvious from any previously disclosed.

Recognition of the importance of the RNAse H sequence specificity and anunderstanding of the reaction mechanism is key to the efficientapplication of this target amplification method to a wide variety oftarget sequences. Moreover, until this time, practitioners assumed thatRNAse H fully and systematically degraded the RNA strand of an RNA:DNAcomplex.

I. The Mechanism of Amplification Methods

Testing amplification efficiency with both prior art methods revealed agreat deal of variability in amplification efficiency with small changesin the primer sets used. The reaction method generally accepted in theprior art did not, in our view, provide a reasonable explanation as towhy one primer set worked so much better than another.

Attempts to improve amplification using the prior art methods did notgive satisfactory results. Efficiency of priming was examined to see ifdifferences in the ability to initiate DNA chains were responsible forobserved differences in primer set efficiency. No correlation betweenpriming efficiency and overall amplification could be found. Analysis ofprimer sets for the ability to form self-complementary structures andcross-complementary structures indicated that differences in primerefficiency were not solely attributable to these factors either.

We also found that the addition of E. coli RNAse H did not uniformlyimprove amplification. As the data submitted herein show, the resultsobserved varied from target to target and from primer set to primer set.The amount of E. coli RNAse H added also is very important and must bekept within a narrowly defined range. With a given target and primerset, addition of E. coli RNAse H is helpful in some cases when thereverse transcriptase is that from avian myeloblastosis virus (“AMV”)but not when the reverse transcriptase is that from Moloney murineleukemia virus (“MMLV”). Data illustrating these conclusions areprovided herein.

Earlier work suggested that AMV reverse transcriptase leaves relativelylarge fragments when it digests the RNA from the RNA:DNA hybrid formedduring virus replication. These fragments serve as primers to initiatesynthesis of the second DNA strand. We report herein our findings thatthere is evidence for sequence specificity of the AMV and MMLV RNAse Hactivities.

In order to elucidate the mechanism of the reaction, individual primerswere terminally labeled with ³²P, and the incorporation of each primerinto DNA products was examined by polyacrylamide gel electrophoresis.According to the generally accepted prior art mechanism, both primerswould be incorporated into full length DNA products. Our experimentsshowed, however, that only the primer complementary to the major RNAspecies synthesized during amplification was incorporated into fulllength product. The primer having the same polarity as the major RNAstrand was never detected in full length DNA product. In fact, itremained quite small. These results were confirmed with a number ofdifferent targets and primer sets.

The failure to detect extension of one of the primers indicated that afully double-stranded DNA intermediate did not accumulate duringamplification, and was not required for autocatalytic amplification.These observations indicate a mechanism for the amplification systems ofthis invention which takes into account probable sequence specificitiesof the RNAse H. The mechanism is generally depicted in FIG. 4.

Experiments have shown that the enzyme cuts the RNA of an RNA:DNA hybridinto specific pieces. Furthermore, the locations of the cut sites wereshown to be in specific regions. To confirm the mechanism, an RNA:DNAhybrid was prepared which contained the plus strand RNA that would begenerated from our T7pro⁺/T7pro⁻ target and primer set combination. The³²P labelled RNA was hybridized to complementary DNA, incubated with AMVreverse transcriptase (containing its associated RNAse H activity), andthe reaction products were analyzed by polyacrylamide gelelectrophoresis (FIG. 5). The fragment size indicated that several smallpieces were generated and that these were produced by cuts near one orboth ends. The interior region of the molecule was not cut. Thisexperiment demonstrates that the enzyme has sequence or structuralspecificity under the reaction conditions used. The results wereentirely consistent with the reaction mechanism of FIG. 4.

Further experiments were performed to determine where the cut sitesoccurred. It is preferred that multiple cut sites occur in the regionhomologous to the promoter-containing primer and not in the regionbinding the other primer. By labeling the termini of the RNAindividually and analyzing the digestion products, it was found thatunder the conditions used the cuts were detected only at the 5′ end ofthe RNA. This is consistent with the mechanism of FIG. 4.

Sequencing experiments were performed to determine the sequences atwhich the RNAse H activities of AMV and MMLV reverse transcriptases cut.Sequences were identified that were specifically cut by each enzyme. Aspredicted, the sequence specificities of the two enzymes are different,and this is believed to explain why some primer sets work better withone enzyme and some with another. The sequence specificities obtainedfor the MMLV enzyme under our reaction conditions do not match thosereported in the literature under another set of reaction conditions,indicating that specificity may be influenced by reaction conditions.

Scrutiny of the role of the RNAse If in the amplification mechanism hasresulted in our finding that completely removing the promoter directedtranscript from its cDNA copy may not be necessary, or even desirablefor formation of a new transcriptionally active template. Thus, in someapplications, even a very low level of RNAse H activity, deriving fromthe reverse transcriptase-intrinsic RNAse H, will be sufficient foreffective amplification if the RNase H is more site selective inallowing the promoter-primer to anneal to the first strand cDNA productor if it interferes less with the annealing of the other primer to thetranscript.

Since E. coli RNAse H is reportedly less specific than the retroviralenzymes, it may cleave in the region to which the non-promotercontaining primer binds, especially if the concentrations of thisprimer, the target, the E. coli RNAse H, and components affecting theenzyme activity are not carefully balanced. In our view these resultsmake the use of E-coli RNase H non-preferable in commercialapplications. Addition of another RNAse H activity, one with differentspecificities, may be useful in those cases in which the reversetranscriptase RNAse H does not cut in the desired regions or does notcut under appropriate conditions. Work with MMLV reverse transcriptase,for example, has shown that this enzyme is less sensitive than the AMVenzyme to inhibition by sample DNA. It is the best mode for manysystems.

New primer sets were designed and are set forth herein based upon themodel and the RNAse H sequence specificity information that we haveobtained to date. Significantly better synthesis was obtained from theseprimer sets than was obtained with those designed previously withoutknowledge of the mechanism and sequence specificities. The inventionherein described makes possible the design of functional primer sets forspecific target regions.

The new mechanism we have discovered involves a novel reactionintermediate for transcription of RNA strands. This intermediate is adouble-stranded complex of RNA and DNA which also contains a doublestranded DNA promoter region. To our knowledge, the reaction isdemonstrably different from any previously disclosed.

An understanding of the reaction mechanism is critical to using thesetarget amplification procedures. Recognition of the importance of theRNAse H sequence specificity is key to the efficient application of thistarget amplification method to a wide variety of target sequences. Onthe other hand, the empirical approach to promoter-primer design is veryintensive, costly, and has a low frequency of success, making thisinvention a useful advance in the art.

A. Narrow Range of Activity for RNAse H Concentration

The amplification system with E. coli RNAse H initially was consideredto be the preferred embodiment because greater synthesis was achievedwith the particular target and primer set being studied, and the E. coliRNAse H was found to be useful in helping to overcome inhibition bysample DNA. However, analysis of the reaction indicates that addition ofE. coli RNAse H is detrimental to amplification in many cases. Moreover,amounts of E. coli RNAse H added must be carefully controlled over anarrow range since the presence of too much or too little is harmful.For practical commercial application of the method, this is asignificant drawback, especially since the enzyme may not be completelystable on storage. In addition, the use of E. coli RNAse H addssignificant cost and complexity to the system. The cost may beprohibitive for many commercial applications. Using E. coli RNAse Hmakes the assay more complex, which in turn increases research anddevelopment costs and may make the assay too delicate for widecommercial application. Thus, our elucidation of the assay mechanism hasresulted in methods for a widely applicable assay both in terms oftechnical feasibility (applicability to target sites) as well as being acheaper and a more robust procedure. Since the addition of E. coli RNAseH was found to result in increased amplification in early experiments,the effect of E. coli RNAse H on the performance of the amplificationsystem in samples containing serum or human DNA was examined in severalexperiments.

Example 11 Optimization of E. coli RNAse H Concentration

Experiments were performed to determine the amount of E. coli RNAse Hneeded for optimal amplification in serum. The following experimentcompared amplification with the T7pro⁺/T7pro⁻ primer pair in thepresence of 0, 0.25, 0.5 and 1 U of RNAse H per assay. HBV+ plasmadiluted to the levels shown in HBV− human serum or HBV− serum alone wastested.

Ten μl of serum were added to an equal volume of 0.1 N KOH and coveredwith a layer of oil to prevent evaporation. The samples were mixed,heated at 95° C. and allowed to cool to room temperature. The sampleswere brought to 90 μl reaction volume with a final concentration of 50mM Tris acetate pH 7.6, 20.8 mM MgCl₂, 5 mM dithiothreitol, 2 mMspermidine hydrochloride, 0.15 μM each primer, 6.25 mM GTP, 6.25 mM ATP,2.5 mM UTP, 2.5 mM CTP, 0.2 mM each dTTP, dATP, dGTP, dCTP and 13 U ofAMV reverse transcriptase. The samples were mixed and heated at 37° C.for 12 minutes, then heated to 95° C. and cooled to room temperature.Thirteen units of RT and 100 U of T7 RNA polymerase were added and thereactions were incubated for three hours at 37° C. Twenty-five μl ofeach reaction was assayed.

The data show that there is a narrow optimum range of concentration ofE. coli RNAse H centering around 0.25 U E. coli RNAse H per reaction forthis system. Even though E. coli RNAse H is difficult to use, some addedRNAse H activity was beneficial in this experiment.

TABLE 11 RNAseH Moles Target (Units) RLU observed 5 × 10⁻²⁰ 0 567809 5 ×10⁻²² 18041 5 × 10⁻²³ 2938 0 1634 5 × 10⁻²⁰ 0.25 1153366 5 × 10⁻²²732109 5 × 10⁻²³ 5566 0 1423 5 × 10⁻²⁰ 0.5 1001904 5 × 10⁻²² 29596 5 ×10⁻²³ 1793 0 1898 5 × 10⁻²⁰ 1.0 610485 5 × 10⁻²² 13026 5 × 10⁻²³ 4062 01662

Example 12

Next the amount of E. coli RNase H needed for optimal amplification ofan HIV primer pair was determined in the presence or absence of a lysatecontaining 8 μg of human DNA. 2×10⁻¹⁸ moles of viral target were presentin each reaction. DNA target was mixed with 50 pmol of each primer in 40mM Tris HCl pH 8.3, 25 mM NaCl, 20.8 mM MgCl₂, 5 mM dithiothreitol, 2 mMspermidine hydrochloride, and triphosphates as described for Table 11,heated to 95° C. and cooled to room temperature. Thirteen units of AMVreverse transcriptase were added and the reaction heated to 42° C. for12 minutes, to 95° C. and cooled again to room temperature. Thirteenunits of AMV reverse transcriptase and 100 units of T7 RNA polymerasewere added and the reactions heated to 37° C. for 3.5 hours prior toassay.

TABLE 12 Lysate RNAse H RLU − 0 U 8,400 − 0.5 239,000 − 1.0 468,000 −1.5 498,000 − 2.0 439,000 − 3.0 20,100 − 4.0 5,806 + .0 1,667 + 0.5924 + 1.0 6,635 + 1.5 579 + 2.0 13,400 + 3.0 17,800 + 4.0 9,152

These results illustrate that E. coli RNAse l levels have to becarefully controlled as too much E. coli RNAse H was detrimental to theamplification. Additionally, the optimal concentration was altered bythe presence of non-specific human DNA and the inhibition by human DNAwas significant at all RNAse H levels.

Example 13

We investigated the effect of E. coli RNAse H on amplification of asecond region referred to as HIV region 2. The following datademonstrate that E. coli RNAse H enhances amplification within a narrowconcentration range. The HIV region 2 primers were amplified asdescribed for Table 12 in the presence of different concentrations of E.coli RNAse H. Ten microliters of each reaction were assayed anddilutions were made when necessary. Signals were compared to a standardcurve to determine the amount of target made.

RNAse H pmole target pmole product Amplification observed

— 0 0 0 — 1.67 × 10⁻¹⁰ 2.14 1.3 × 10¹⁰ 0.2 U 1.67 × 10⁻¹⁰ 0.17 1.0 × 10⁹0.4 U 1.67 × 10⁻¹⁰ 0.18 1.1 × 10⁹ 1.2 U 1.67 × 10⁻¹⁰ 0.012 7.6 × 10⁷ —1.67 × 10⁻⁸ 16.0 9.6 × 10⁸ 0.2 U 1.67 × 10⁻⁸ 20.6 1.2 × 10⁹ 0.4 U 1.67 ×10⁻⁸ 0.14 8.5 × 10⁶ 1.2 U 1.67 × 10⁻⁸ 0.15 9.0 × 10⁶

These data show that amplification can be achieved without the additionof E. coli RNAse H, contrary to the assertions of Guatelli, et al., 87PNAS 1874-1878.

We investigated using E. coli RNAse H with MMLV reverse transcriptase inseveral target regions. Reactions were done in the presence or absenceof 8 μg human DNA using conditions described for Example 12, with a 3hour autocatalysis step. Primer sets from HIV regions 1, 3 and 4 weretested. The amount of viral template used was selected to give RLU inthe linear range of the hybridization assay. MMLV reverse transcriptasewas used at 400 U during initiation, 800 U for efficient amplification.400 U of T7 RNA polymerase were included, and 1 U of E. coli RNAse H wasadded as indicated. Values presented under the column headings, +RNAseH, −RNAse H, are RLUs obtained from assay of 10 μl of the reactions.

TABLE 15 Target Human Region Moles Target DNA +RNAse H −RNAse H HIVregion 1 2 × 10⁻²¹ — 54,900 137,000 HIV region 1 2 × 10⁻²¹ 8 μg 15,10013,800 HIV region 3 2 × 10⁻²⁰ — 96,100 391,000 HIV region 3 2 × 10⁻²⁰ 8μg 124,000 246,000 HIV region 4 2 × 10⁻²¹ — 20,400 107,000 HIV region 42 × 10⁻²¹ 8 μg 56,000 8,800

In the presence of DNA, E. coli RNAse H apparently stimulatedamplification directed by the HIV region 4 primers. In most cases wehave tested, amplification using MMLV reverse transcriptase alone is atleast as good as when MMLV reverse transcriptase is used with E. coliRNAse H. E. coli RNAse H is not required for efficient amplification,contrary to the assertions of Guatelli, et al., 87 PNAS 1874-1878.

Sequence Specificity of Reverse Transcriptase

We also have discovered that some primer sets work best with AMV reversetranscriptase while others work best with MMLV reverse transcriptase orwith one of the reverse transcriptases and added E. coli RNAse H. Theobserved degree of variability in amplification efficiency with smallchanges in promoter primers and primers or source of RNAse H supportsour proposed mechanism. We set forth below our detailed data in supportof these findings.

MMLV reverse transcriptase has been cloned, and is commerciallyavailable from BRL (Bethesda Research Labs), U.S. Biochemicals andothers in a very concentrated form (greater than 300 units per μl). Itshould be noted that comparable DNA synthetic activity on naturalnucleic acid templates is obtained with approximately 10-fold greaterunit concentration of MMLV reverse transcriptase compared to AMV reversetranscriptase. Lack of comparability in unit activity is due to the factthat the enzymes show different relative activities when tested withhomopolymer templates (used in the unit activity assay) andheteropolymeric nucleic acid templates. We tested the use of MMLVreverse transcriptase at various levels in our amplification reactions.Examples of these results are shown in the tables below. In the AMVreverse transcriptase samples, reverse transcriptase was used at 14 Uduring the initiation step and 56 U during the amplification step. Theamount of MMLV reverse transcriptase was titrated for both theinitiation and the amplification steps. The incubation conditions usedwere as described for Example 12 except that 15 pmol of each HIV region2 primer was used and 25 mM KCl replaced the NaCl. T7 polymerase wasused at 400 U during the amplification. The following table showsperformance in the presence or absence of 8 μg human DNA. Columns headedwith the designation AMV or MMLV show the results of amplificationsperformed with AMV reverse transcriptase or MMLV reverse transcriptase,respectively. The numbers refer to the number of units used duringinitiation and autocatalysis, respectively. The values contained withinthe table are RLUs. Note that dilutions of the amplification productswere not performed and values >200,000 RLU may significantlyunderestimate the extent of amplification since signal saturation occursat a level of hybridization target sufficient to give about 250,000 RLUwith the conditions used.

TABLE 16 Human AMV MMLV MMLV MMLV Moles Target DNA 14/56 400/400 400/600400/800 0 − 495 470 — 3,800 1.6 × 10⁻²² − 278,000 77,000 — 5,621 1.6 ×10⁻²⁰ − 292,000 276,000 — 269,000 0 + 474 547 488 1,352 1.6 × 10⁻²⁰ +10,200 62,700 205,000 149,000

Although the sensitivity of amplification directed by MMLV reversetranscriptase in the absence of human DNA was significantly lower thanAMV directed amplification, the MMLV was much more effective in thepresence of exogenous DNA.

After observing the high level amplification of the HIV region 2, wetested the other target regions in the presence of human DNA and foundthat, using AMV reverse transcriptase, E. coli RNAse H was stillrequired for the most effective amplification in these regions. We thentested each target region using MMLV reverse transcriptase, without E.coli RNAse H, to compare amplification performance with reactionscontaining AMV reverse transcriptase+E. coli RNAse H. An example ofthese results for two target regions is shown in the table below. TheHIV region 3 and 4 primers were used (50 pmol per reaction) as describedfor Example 12. In reactions using AMV reverse transcriptase, 14 U wasused at initiation, 56 U reverse transcriptase+1 U E. coli RNAse H wereadded for amplification. In reactions using MMLV reverse transcriptase,400 U was added at initiation and 800 U for amplification. All reactionscontained 400 U T7 RNA polymerase during the four-hour amplificationincubation. Values within the tables are RLU obtained from HomogeneousProtection Assay performed using 10 μl of the amplification reactions.Dilutions of the reactions were performed in some cases before assay.

TABLE 17 Human HIV region 3 HIV region 4 Moles Target DNA AMV MMLV AMVMMLV 0 − 2,049 751 1,184 777 1.6 × 10⁻²¹ − 70,800 689 2.1 × 10⁷ 305,0001.6 × 10⁻²⁰ − 510,000 1,869 — — 0 + 551 400 1,058 1,182 1.6 × 10⁻²¹ + —— 13,900 16,200 1.6 × 10⁻²⁰ + 706 1,862 141,000 154,000 1.6 × 10⁻¹⁹ + —— 683,000 723,000 1.6 × 10⁻¹⁸ + 10,800 115,000 — —

As observed in the HIV region 2, in the absence of human DNA, theamplification with MMLV is significantly less than with AMV reversetranscriptase with E. coli RNAse H but in the presence of DNA the MMLVdirected amplification is at least as good as accomplished by AMVreverse transcriptase with RNAse H.

Example 14 Primers for Second Region of HBV Genome

The following experiment was performed with primer sets directed to tworegions of the HBV genome. The data show that the primer sets do notamplify to the same extent with AMV RT. The experiment was performed asdescribed for Example 11 using. HBV positive plasma diluted in negativeserum. Ten microliters of amplification reaction were tested in thehybridization assay.

TABLE 18 RLU observed Moles Target HBV Region 1 HBV Region 2 4.8 × 10⁻²¹690,674 4.8 × 10⁻²² 475,849 73,114 4.8 × 10⁻²³ 242,452 4,193 0 1,4171,940

These results were confirmed by additional experiments using standardprotocols. The region 1 primers consistently gave higher RLU in theseexperiments.

In contrast, when 800 U of MMLV enzyme were used to amplify the same twoprimer pairs, the opposite effect was seen as shown below.

TABLE 19 RLU observed Moles Target Region 1 Region 2 9.6 × 10⁻²⁰ 37,2781,067,951 9.6 × 10⁻²² 1,858 40,826 0 1,010 1,646

In this experiment, each reaction contained 5 μl serum.

Thus, the amplification potential of each primer pair was influenced bythe reverse transcriptase present during amplification. Factors such asthe availability of the template sequences, ability of the primers tohybridize specifically, and efficiency of RNA synthesis should not beaffected significantly by the type of reverse transcriptase present.Factors such as RNAse H specificity and activity and DNA polymerizingactivity could be affected.

The following data illustrates that promoter-primer combinations used inthe appropriate conditions can be designed to increase amplification.

TABLE 21 This experiment was performed with HBV region 2 primers asdescribed for Table 11 except that the entire amplification reaction wasanalyzed by hybridization. Target Molecules Moles Target RLU Observed1200 2 × 10⁻²¹ 1,094,177 120 2 × 10⁻²² 442,137 12 2 × 10⁻²³ 24,053 1.2 2× 10⁻²⁴ 8,654 0 0 1,828

Example 15 Comparison of AMV Reverse Transcriptase and MMLV ReverseTranscriptase

The following experiment compared amplification of primers for a BCL-2chromosonal translocation major human chromosomal breakpoint t(14;18)found in patients with CML using MMLV (300 units) or AMV (39 units). Theeffect of E. coli RNAse H was evaluated with each enzyme. Amplificationswere performed as described for Example 12 except that 24 mM MgCl₂ and50 pmol each primer were used. In reactions containing lysate, 4 μg ofDNA from white blood cells was present. All reactions contained 300units T7 RNA polymerase and 10 amol of input double-stranded DNA target.

TABLE 22 0 Units 0.5 Units 1.0 Units 2.0 Units RT Lysate RNAse H RNAse HRNAse H RNAse H AMV − 108,018 2,035,377 — — + 44,485 204,894 165,972136,647 MMLV − 3,015,948 2,224,666 — — + 3,070,136 2,714,257 767,218105,845

The results show that MMLV and AMV RT do not amplify this primer set tothe same extent, particularly in the absence of E. coli RNAse H. E. coliRNAse H added to reactions containing AMV reverse transcriptase markedlyimproved amplification; this indicates that the RNAse activity waslimiting in these reactions. In contrast, E. coli RNAse H did notenhance amplification when added to reactions containing MMLV RT. Thedata also confirms a point already made concerning the ability of MMLVRT to sustain significant amplification in the presence of large amountsof nonspecific human DNA.

C. One Primer Was Not Incorporated into Full Length Product

One of our most important findings is that the primer of the samepolarity as the major RNA species was not detectably incorporated intofull length DNA product. To demonstrate this, individual primers wereterminally labeled with ³²P, and the incorporation of each primer intoDNA products was examined by polyacrylamide gel electrophoresis. Weinitially expected both primers to be incorporated into full length DNAproducts. However, the primer containing the promoter was not observedin full length DNA product. In fact, it remained quite small. Theseresults were confirmed with a number of different targets and primersets. Our method is explained below.

To identify the species of cDNA accumulated during autocatalysis,primers were ³²P-labeled at the 5′ end with T4 polynucleotide kinase andspiked into amplification reactions. The following examples show thatcDNA of one polarity is preferentially accumulated during amplification,and that the accumulated strand is always complementary to thepredominant RNA species synthesized during autocatalysis. FIG. 5 showsthe result of incorporation of ³²P-labeled HIV region 2 primers duringamplification. This primer set results in synthesis of a 214 base RNA ofthe (+) sense. The primer complementary to the RNA was incorporated intotwo major bands of 235 and 214 bases when target sequences were present(lane 3). No full length -fragments were seen in the absence of target(lane 4). The 214 base fragment represents cDNA equal in length to theRNA while the 235 base fragment is equal in length to the RNA+21 basesof the T7 promoter sequence. In contrast, the promoter primer was notobserved in full length DNA product in the presence or absence of target(lanes 1 and 2 respectively).

Lanes 5-8 of FIG. 5 show the result of incorporation of ³²P-labeled HBVregion 1 primers during amplification. These are known as T7pro⁺ andT7pro⁻. This primer set is capable of producing 132 base RNA of twopolarities but the (+) strand RNA predominates. T7pro⁻, which iscomplementary to the predominant RNA, was incorporated into fragments of132 bases and 153 bases consistent with our proposed mechanism (lane 7(+) target, lane 8, (−) target). The 153 base fragment is equal inlength to the RNA+21 bases of the T7 promoter sequence of the T7 pro⁺.In contrast, ³²P-labeled T7pro⁺ primer was not incorporated intofragments of either length (lane 5 (+) target, lane 6, (−) target).

The reactions analyzed by gel electrophoresis were also analyzed by HPAto determine if cDNA of the same polarity as the predominant RNA couldbe detected by another method. Plus and minus strand probes were used todetermine the relative ratio of strands made, and a portion of eachreaction was treated with RNAse A to determine the ratio of RNA to DNA.The results are set forth below.

TABLE 23 RLU RLU Probe Polarity Printer set No treatment RNase A (−)*HIV Region 2 679,182 1037 HIV Region 2 453,675 1464 (+) *HIV Region 232,922 1,094,249 HIV Region 2 39,494 655,595 (−) *HBV Region 1 567,1104,854 HBV Region 1 671,656 4,210 (+) *HBV Region 1 56,550 303,160 HBVRegion 1 77,983 450,332 * = (+) strand primer labeled with ³²P, others,(−) strand primer labeled with ³²P.

These results show that the amplifications worked well, even when fulllength product was not observed with the promoter primer. These resultscorrelate with what was observed in the previous study, that is, most ofthe signal observed with one sense probe is from RNA, and thecomplementary strand signal is as expected, from DNA. This was true evenfor the HBV region 1 primer set which should have made RNA of bothpolarities.

D. Confirmation of Mechanism Showing that the Enzyme Cuts RNA of theRNA/DNA Hybrid at Specific Loci

Based upon the experiments and observations reported hereinabove, themechanism for the amplification systems that takes into account probablesequence specificities in the RNAse H is depicted in FIG. 4. In supportof this mechanism, since RNAse H sequence specificity is a key element,it is necessary to show that indeed the enzymes cut the RNA of anRNA:DNA hybrid at specific locations. Furthermore, the locations of thecut sites needed to be in specific regions according to the model inorder for good amplification to be obtained. To examine this question,an RNA:DNA hybrid was prepared that contained the RNA that would begenerated from a known target and primer set combination. The RNA waslabeled with ³²P, incubated with AMV reverse transcriptase (containingits associated RNAse H activity) and the reaction products were analyzedby polycryamide gel electrophoresis. The results were entirelyconsistent with the new reaction mechanism, namely, the fragment sizeindicated that several small pieces were generated and that these wereproduced by cuts near one or both ends. The interior region of themolecule was not cut. This experiment confirmed that the enzyme hassequence or structural specificity under the reaction conditions used.

Further experiments were performed to determine where the cuts occurredsince the proposed mechanism requires that multiple cuts occur in theregion binding the promoter-containing primer. By labeling the terminiof the RNA individually and analyzing the digestion products, it wasdemonstrated that the cuts were made only at the 5′ end of the RNA. Thisalso is consistent with the proposed mechanism.

Example 16

FIG. 6 shows that the RNAse H activities from AMV, MMLV and E. coli cutat specific RNAse H cleavage sites. The arrows in the figure indicatethe position of full-length RNA.

FIG. 6 shows the result of an experiment in which HIV region 2 RNA wasinternally labelled with ³²P, hybridized to a single-stranded targetsequence and nicked with RNAse H from AMV for 45 minutes (lane 2), MMLVfor 5, 15, 45 or 120 minutes (lane 3-6) or E. coli RNAse H for 5 minutes(lane 7). The sizes of fragments produced were discrete and differentwith each enzyme, indicating specificity of cleavage of the enzymes andvaried specificity among enzymes with this template. The most rapiddegradation was observed in the presence of E. coli RNAse H, indicatingless specificity or greater activity of cutting with this enzyme.

FIG. 6b shows the results of hybridization of HBV region 1 RNA to asynthetic target sequence, followed by nicking with RNAse H from AMVreverse transcriptase for 5, 30, 60 or 120 minutes (lanes 2-5) or E.coli for 1, 3, 15 or 60 minutes (lanes 6-9). Different sized fragmentswere produced with the two enzymes, indicating specificity of cleavage.When the HBV RNA was labeled on the 3′ terminus and nicked with AMVreverse transcriptase, the same sized fragments were observed,indicating that the cleavage sites were near the 5′ end of the RNA.

These data indicate that specific sites are cleaved with RNAse H fromAMV, MMLV and E. coli, and that at least some sites are different withthe three enzymes. The presence of specific sites within the region tobe amplified allows the RNA in an RNA:DNA hybrid to be cleaved, allowingautocatalysis to occur efficiently. Primers designed using cut siteinformation show improved amplification efficiency. This is consistentwith our observations that certain primer sets amplified to differentextents depending on the source of RNAse H.

E. Identification of MMLV and AMV RNAse H Cut Sites

Example 17

To identify sites digested by AMV RNAse H, RNA was hybridized with acomplementary sequence and nicked with AMV RNAse H. Followingdenaturation, a primer complementary to the 3′ end of the region to besequenced was hybridized and extended in the presence ofdideoxynucleotides by the Sanger sequencing method. Termination of cDNAsynthesis, indicating cleavage of RNA, was observed at the followingsites for the HBV RNA:

5′.GGGAGAGGUUAUCGC*UGGA*UGUGUCUGCGGCGUUUUAUCA*UAUUCCUCUUCA*UCCUG . . .3′ (SEQ ID NO:32).

To identify sites digested by MMLV RNAse H, RNA was hybridized with acomplementary sequence and nicked with MMLV RNAse H. Followingdenaturation, a primer complementary to the 3′ end of the region to besequenced was hybridized and extended in the presence ofdideoxynucleotides by the Sanger sequencing method. Termination of cDNAsynthesis was observed at the following sites for the HBV RNA:

5′.GGGAGAGGUUAUCGC*UGGA*UGUGUCUGCGGC*GUUUUAUCA*UAUUCCUCUUCAUCCUGC*UGCUAUGCCUCA*UCUUC. . . -3′ (SEQ ID NO:34).

The following sites were identified for a second HBV RNA sequence:

5′.GGGAGACCCGAGAU*UGA*GAUCUUCUGCGACGCGGCGAU*UGA*GAUCUGCGUCU*GCGAGGCGAGGGAGU*UCU*UCUU*CUAGGGGACCUGCCUCGGUCCCGUC*GUCUA. . . 3′ (SEQ ID NO:35).

The following sites were identified for an HIV RNA sequence:

5′.GGGAGACAAA*UGGCAGUA*UUCAUCCACAAUUUUAAAAGAAAAGGGGGGAUUGGGGGGUACAGUGCAGGGGAAAGAAUAGUAGACAUAAUAGC*AACAGACAUAC*AAACUAAAGAAUUACAAAAACAAAUUAC*AAAAAUUCAAAAUUUUCGGGUUUAUUACAGGGAC*AGC*AGAAA. . . 3′ (SEQ ID NO:36).

Most of the cleavage sites occurred near the dinucleotides CA or UG. Themethod used for detecting cleavage sites only identified sites whichaccumulated during the cleavage reaction. It is expected that additionalsites could be cleaved which were not recognized by the method used.

F. Primers for Amplification Systems

Based on findings that the various RNAse H enzymes have sequencespecificity, we have tested various primer/target combinations andattempted to optimize their performance in amplification systems. Dataobtained to date indicates that the piece size of the RNA fragmentsproduced is relatively large and that the fragments probably do notspontaneously dissociate from the duplex. This is not unexpected sincework with AMV reverse transcriptase copying AMV RNA or poliovirus RNAshowed that the RNA fragments that were produced by the RNAse H wereused by the enzyme to prime the synthesis of cDNA from the initiallysynthesized cDNA strand.

If the RNAse H enzymes have sequence specificity, the amplificationreaction proceeds as follows (beginning with the RNA intermediate in thereaction):

The primer complementary to the major RNA species produced duringamplification binds at the 3′ terminus of the RNA. Since theconcentration of primer is high in the reaction, excess primer producesRNA:DNA duplexes which may be cut by the RNAse H activity before beingable to initiate synthesis. Therefore, it is preferable that the primerbinding region does not contain a large number of sequences recognizedby the RNAse H enzyme used in the reaction.

As cut sites occur frequently, it may not be practical in some cases todesign an RNA complementary primer without recognized cut sites; in suchcases, the cut sites should be as near the 5′ terminus as possible toallow the 3′ terminal portion of the primer to remain annealed to theRNA.

Upon extension of the primer by a suitable DNA polymerase, the bindingsite for the second primer, which contains the RNA polymerase promoter,must now be exposed. It is sufficient to remove only a portion of theRNA to allow nucleation and zippering of the primer as it hybridizes tothe cDNA, to allow reverse transcriptase mediated binding of the primerand initiation of synthesis, or merely to nick the RNA so that the RNAfragment that results may be displaced. Since our data show relativelylarge pieces of RNA are made and that the promoter containing primer isnot incorporated into full-length DNA, the following events can occur:

1. There is sufficient nicking of the RNA to permit binding of thepromoter-primer. Whether a nick in the appropriate place simply producesan RNA fragment sufficiently small to melt off and expose the primerbinding site or a portion thereof or whether a nick allows an unwindingactivity associated with one or more of the enzymes to displace the RNAfragment is not known at this time.

2. The cDNA 3′ terminus is extended by the reverse transcriptase to makethe promoter region double-stranded DNA.

3. RNA is synthesized from the complex thus made. This complex wouldconsist of a cDNA bound to RNA and containing a double-stranded DNApromoter.

Thus, there must be a sequence recognized by the RNAse H activitypresent in the reaction somewhere in or near the binding site for theprimer containing the RNA polymerase promoter.

In some applications, it may also be desirable to not have RNAse Hrecognition sites within the target sequence itself. Sites within thetarget may be cleaved and used to produce RNA primers for synthesis ofdouble-stranded cDNA regions. It may be preferable to eliminate thepossibility of this enzymatic activity.

New primer sets were designed based upon the model and the RNAse Hsequence specificity information that we have obtained. Our designcriteria are as follows:

For the T7 Promoter-primer:

1) The primer region should have one or more cut sites per 10 bases.

2) The primer region should have a cut site near the 5′ end.

3) The primer region should have a cut site near the 3′ end and possiblya partial site at the 3′ end.

4) The primer length should be >18 bases.

5) The T_(m) estimated should be about 55-65° C.

For the other primer:

1) The primer should have few or no RNAse H cut sites.

2) Any cut sites in the primer should be near the 5′ end.

3) The primer length should be about 18-28 bases in length.

4) The T_(m) estimated should be about 55-65° C.

Significantly better synthesis was obtained from primer sets designedusing these criteria and knowledge of the mechanism and sequencespecificities. This shows the utility of the invention in makingpossible the design of functional primer sets for specific, targetregions. These are explained more fully below.

Example 18

Our findings regarding RNAse H specificity have been used to designefficient promoter-primer combinations. Prior art methods simplynonselectively attached promoters to primer sequences. We have been ableto design and optimize promoter-primer combinations to increase theyield of amplified product. The following experiment shows that smallchanges in promoter-primer sequence result in large changes inamplification efficiency.

The following examples show primers from similar regions which werecompared for RNAse H cleavage sites and GP-III amplification efficiency.In each example, duplicate amplifications were performed using commonreagents except for the primers being tested.

1. Non-promoter Primers

In the first example, the non-promoter primer site for the CML majort(14; 18) breakpoint amplification region was moved 15 bases, resultingin a reduction in the number of putative RNAse H cut sites from 4 to 1,assuming a 4 base recognition sequence or from 5 to 2 assuming a 3 baserecognition sequence. The reaction was performed as described forExample 15 except that 2.5 mM ATP, 16.5 mM MgCl₂ and 50 mM KCl wereincluded. This change in primer sequence had a dramatic positive effecton amplification efficiency. In the second case, an intentional mismatchwas placed internally in the non promoter primer of HBV region 1 toremove the only putative RNAse H cut site, assuming a 4 base recognitionsite. In the case of a 3 base cut site, one skilled in the art wouldrecognize that the mismatch removed the cut site nearest the 3′ end.This change also had a definitive positive effect on amplificationefficiency. The data demonstrate that two methods, changing the positionof the primer, or inclusion of mismatches, can be used to enhanceamplification. Presumably, removal of RNAse H cut sites from thenon-promoter primer results in more efficient priming of cDNA synthesisduring autocatalysis.

Sequence RLU

Example 1

SEQ ID NO:46: GGAGCTGCAGATGCTGACCAAC 78,880

SEQ ID NO:37: GACCAACTCGTGTGTGAAACTCCA 2,552,333

Example 2

SEQ ID NO:33: TCCTGGAATTAGAGGACAAACGGGC 57,710

SEQ ID NO:38: TCCTGGAATTAGAGGATAAACGGGC 518,695

2. Promoter-primers

The following examples show promoter primers which come from similarregions but which differ in the number of putative RNAse H cut sites. Inthe first case, the two promoter primer sites for the HIV region 5 aredisplaced by 10 bases, changing the number of putative RNAse H cut sitesfrom two to three, assuming a four base recognition site, or from 3 to 5assuming a 3 base recognition site. This has a positive effect onamplification efficiency. In the second case, a sequence containingputative RNAse H cut sites was inserted upstream of the promoter primerfor the major breakpoint t(14; 18) translocation, and one mismatch tothe target was included to generate a cut site within the primer region.This also had a positive effect on amplification efficiency. Thisdemonstrates that insertion of RNAse H cut sites in the promoter primercan be used to enhance amplification efficiency. Presumably, inclusionof RNAse H cut sites assists in RNA strand displacement, increasing theefficient of copying of the promoter region, thereby resulting in moreefficient autocatalysis.

Primer name RLU Example 3 A 45,466 B 908,147 Example 4 C 64,601 D2,946,706

Sequences of the primers above are:

Primer A:

AATTTTAATACGACTCACTATAGGGAGAAATCTTGTGGGGTGGCTCCTTCT-3′ (SEQ ID NO: 16)

Primer B:

AATTTAATACGACTCACTATAGGGAGAGAGGGGTGGCTCCTTCTGATAATGCTG-3′ (SEQ ID NO:15)

Primer C:

ATTTAATACGACTCACTATAGGGAGACGGTGACCGTGGTCCCTTG-3′ (SEQ ID NO:39)

Primer D:

TAAATTAATACGACTCACTATAGGGAGATCAGTTACAATCGCTGGTATCAACGCTGAGCAGACGCTGACCGTGGTCCCTTG-3′(SEQ ID NO:40).

In the above examples, removal of RNAse H cut sites from thenon-promoter primer resulted in enhanced amplification, even if theremoval of the cut site involved the incorporation of a mismatch to theoriginal target. Design of the promoter-containing primer to includeadditional RNAse H cut sites also enhanced amplification, again, even ifthe incorporation of cut sites involved inclusion of mismatches to theoriginal target. The number, distribution, and position of putativeRNAse H cut sites determine, in part, the usefulness of a given primer.

Improvement of amplification by inclusion of intentional mismatches orinsertion of sequences between the promoter and primer are nonobviousimprovements to the amplification method.

In a preferred embodiment of the present invention, the RNA targetsequence is determined and then analyzed to determine where RNAse Hdegradation will cause cuts or removal of sections of RNA from theduplex. Experiments can be conducted to determine the effect of theRNAse degradation of the target sequence by RNAse H present in AMVreverse transcriptase and MMLV reverse transcriptase, by E. coli RNAse Hor by combinations thereof.

In selecting a primer, it is preferable that the primer be selected sothat it will hybridize to a section of RNA which is substantiallynondegraded by the RNAse H present in the reaction mixture. If there issubstantial degradation, the cuts in the RNA strand in the region of theprimer may stop or inhibit DNA synthesis and prevent extension of theprimer. Thus, it is desirable to select a primer which will hybridizewith a sequence of the RNA target, located so that when the RNA issubjected to RNAse H, there is no substantial degradation which wouldprevent formation of the primer extension product.

The site for hybridization of the promoter-primer is chosen so thatsufficient degradation of the RNA strand occurs to permit removal of theportion of the RNA strand hybridized to the portion of the DNA strand towhich the promoter-primer will hybridize. Typically, only portions ofRNA are removed from the RNA:DNA duplex by RNAse H degradation and asubstantial part of the RNA strand remains in the duplex. An RNA:DNAduplex containing a double-stranded DNA promoter results.

46 1 28 DNA Artificial Sequence Synthesized nucleic acid molecule 1gcagctgctt atatgcagga tctgaggg 28 2 54 DNA Artificial SequenceSynthesized nucleic acid molecule 2 aatttaatac gactcactat agggagacaagggactttcc gctggggact ttcc 54 3 27 DNA Artificial Sequence Synthesizednucleic acid molecule 3 gtctaaccag agagacccag tacaggc 27 4 29 DNAArtificial Sequence Synthesized nucleic acid molecule 4 ctactattctttcccctgca ctgtacccc 29 5 49 DNA Artificial Sequence Synthesized nucleicacid molecule 5 aatttaatac gactcactat agggagacaa atggcagtat tcatccaca 496 18 DNA Artificial Sequence Synthesized nucleic acid molecule 6cccttcacct ttccagag 18 7 31 DNA Artificial Sequence Synthesized nucleicacid molecule 7 gactagcgga ggctagaagg agagagatgg g 31 8 24 DNAArtificial Sequence Synthesized nucleic acid molecule 8 ctcgacgcaggactcggctt gctg 24 9 50 DNA Artificial Sequence Synthesized nucleic acidmolecule 9 aatttaatac gactcactat agggagactc ccccgcttaa tactgacgct 50 1026 DNA Artificial Sequence Synthesized nucleic acid molecule 10cttccccttg gttctctcat ctggcc 26 11 54 DNA Artificial SequenceSynthesized nucleic acid molecule 11 aatttaatac gactcactat agggagagaccatcaatgag gaagctgcag aatg 54 12 26 DNA Artificial Sequence Synthesizednucleic acid molecule 12 ccatcctatt tgttcctgaa gggtac 26 13 31 DNAArtificial Sequence Synthesized nucleic acid molecule 13 gaaggctttcagcccagaag taatacccat g 31 14 31 DNA Artificial Sequence Synthesizednucleic acid molecule 14 ggcaaatggt acatcaggcc atatcaccta g 31 15 52 DNAArtificial Sequence Synthesized nucleic acid molecule 15 aatttaatacgactcactat agggagaggg gtggctcctt ctgataatgc tg 52 16 51 DNA ArtificialSequence Synthesized nucleic acid molecule 16 aattttaata cgactcactatagggagaaa tcttgtgggg tggctccttc t 51 17 27 DNA Artificial SequenceSynthesized nucleic acid molecule 17 caagggactt tccgctgggg actttcc 27 1822 DNA Artificial Sequence Synthesized nucleic acid molecule 18caaatggcag tattcatcca ca 22 19 23 DNA Artificial Sequence Synthesizednucleic acid molecule 19 ctcccccgct taatactgac gct 23 20 25 DNAArtificial Sequence Synthesized nucleic acid molecule 20 ccatcaatgaggaagctgca gaatg 25 21 25 DNA Artificial Sequence Synthesized nucleicacid molecule 21 ggggtggctc cttctgataa tgctg 25 22 24 DNA ArtificialSequence Synthesized nucleic acid molecule 22 aaatcttgtg gggtggctcc ttct24 23 26 DNA Artificial Sequence Synthesized nucleic acid molecule 23ggtcccctag aagaagaact ccctcg 26 24 33 DNA Artificial SequenceSynthesized nucleic acid molecule 24 caccaaatgc ccctatctta tcaacacttccgg 33 25 51 DNA Artificial Sequence Synthesized nucleic acid molecule25 aatttaatac gactcactat agggagaccc gagattgaga tcttctgcga c 51 26 54 DNAArtificial Sequence Synthesized nucleic acid molecule 26 aatttaatacgactcactat agggagaggt tatcgctgga tgtgtctgcg gcgt 54 27 25 DNA ArtificialSequence Synthesized nucleic acid molecule 27 gaggacaaac gggcaacataccttg 25 28 52 DNA Artificial Sequence Synthesized nucleic acid molecule28 aatttaatac gactcactat agggagatcc tggaattaga ggacaaacgg gc 52 29 25DNA Artificial Sequence Synthesized nucleic acid molecule 29 cctcttcatcctgctgctat gcctc 25 30 24 DNA Artificial Sequence Synthesized nucleicacid molecule 30 gagcatagca gcaggatgaa gagg 24 31 25 DNA ArtificialSequence Synthesized nucleic acid molecule 31 gcagagttca aaagcccttcagcgg 25 32 57 DNA Artificial Sequence Synthesized nucleic acid molecule32 gggagagguu aucgcuggau gugucugcgg cguuuuauca uauuccucuu cauccug 57 3325 DNA Artificial Sequence Synthesized nucleic acid molecule 33tcctggaatt agaggacaaa cgggc 25 34 75 DNA Artificial Sequence Synthesizednucleic acid molecule 34 gggagagguu aucgcuggau gugucugcgg cguuuuaucauauuccucuu cauccugcug 60 cuaugccuca ucuuc 75 35 104 DNA ArtificialSequence Synthesized nucleic acid molecule 35 gggagacccg agauugagaucuucugcgac gcggcgauug agaucugcgu cugcgaggcg 60 agggaguucu ucuucuaggggaccugccuc ggucccgucg ucua 104 36 173 DNA Artificial SequenceSynthesized nucleic acid molecule 36 gggagacaaa uggcaguauu cauccacaauuuuaaaagaa aaggggggau ugggggguac 60 agugcagggg aaagaauagu agacauaauagcaacagaca uacaaacuaa agaauuacaa 120 aaacaaauua caaaaauuca aaauuuucggguuuauuaca gggacagcag aaa 173 37 24 DNA Artificial Sequence Synthesizednucleic acid molecule 37 gaccaactcg tgtgtgaaac tcca 24 38 25 DNAArtificial Sequence Synthesized nucleic acid molecule 38 tcctggaattagaggataaa cgggc 25 39 45 DNA Artificial Sequence Synthesized nucleicacid molecule 39 atttaatacg actcactata gggagacggt gaccgtggtc ccttg 45 4081 DNA Artificial Sequence Synthesized nucleic acid molecule 40taaattaata cgactcacta tagggagatc agttacaatc gctggtatca acgctgagca 60gacgctgacc gtggtccctt g 81 41 45 DNA Artificial Sequence Synthesizednucleic acid molecule 41 gaattaatac gactcactat agggagacct gaggagacggtgacc 45 42 20 DNA Artificial Sequence Synthesized nucleic acid molecule42 tatggtggtt tgacctttag 20 43 24 DNA Artificial Sequence Synthesizednucleic acid molecule 43 ggctttctca tggctgtcct tcag 24 44 24 DNAArtificial Sequence Synthesized nucleic acid molecule 44 ggtcttcctgaaatgcagtg gtcg 24 45 50 DNA Artificial Sequence Synthesized nucleicacid molecule 45 gaattaatac gactcactat agggagactc agaccctgag gctcaaagtc50 46 22 DNA Artificial Sequence Synthesized nucleic acid molecule 46ggagctgcag atgctgacca ac 22

What is claimed is:
 1. An oligonucleotide probe, wherein the basesequence of said probe consists of or is contained within a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:13, and the sequences perfectlycomplementary thereto, wherein said probe hybridizes to HIV nucleic acidin a sample to a form a detectable probe:target duplex which indicatesthe presence of HIV nucleic acid under hybridization conditions, andwherein said probe does not hybridize to human nucleic acid in saidsample to form a detectable probe:non-target duplex under saidconditions.
 2. The probe of claim 1, wherein the base sequence of saidprobe consists of or is contained within the sequence of SEQ ID NO:1 orthe complement thereof.
 3. The probe of claim 2, wherein the basesequence of said probe consists of the sequence of SEQ ID NO:1 or thecomplement thereof.
 4. The probe of claim 1, wherein the base sequenceof said probe consists of or is contained within the sequence of SEQ IDNO:4 or the complement thereof.
 5. The probe of claim 4, wherein thebase sequence of said probe consists of the sequence of SEQ ID NO:4 orthe complement thereof.
 6. The probe of claim 1, wherein the basesequence of said probe consists of or is contained within the sequenceof SEQ ID NO:7 or the complement thereof.
 7. The probe of claim 6,wherein the base sequence of said probe consists of the sequence of SEQID NO:7 or the complement thereof.
 8. The probe of claim 1, wherein thebase sequence of said probe consists of or is contained within thesequence of SEQ ID NO:10 or the complement thereof.
 9. The probe ofclaim 8, wherein the base sequence of said probe consists of thesequence of SEQ ID NO:10 or the complement thereof.
 10. The probe ofclaim 1, wherein the base sequence of said probe consists of or iscontained within the sequence of SEQ ID NO:13 or the complement thereof.11. The probe of claim 10, wherein the base sequence of said probeconsists of the sequence of SEQ ID NO:13 or the complement thereof. 12.The probe of claim 1, wherein said probe includes a detectable label.13. A kit for detecting whether HIV nucleic acid may be present in asample comprising: a) an oligonucleotide probe, wherein the basesequence of said probe consists of or is contained within a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:13, and the sequences perfectlycomplementary thereto, wherein said probe hybridizes to HIV nucleic acidin a sample to a form a detectable probe:target duplex which indicatesthe presence of HIV nucleic acid under hybridization conditions, andwherein said probe does not hybridize to human nucleic acid in saidsample to form a detectable probe:non-target duplex under saidconditions; and b) one or more oligonucleotide primers, wherein the basesequence of each of said primers consists of or is contained within asequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and thesequences perfectly complementary thereto.
 14. The kit of claim 13,wherein said primers include first and second primers, and wherein: a)the base sequence of said probe consists of or is contained within thesequence of SEQ ID NO:1; b) the base sequence of said first primerconsists of or is contained within the sequence of SEQ ID NO:2; and c)the base sequence of said second primer consists of or is containedwithin the sequence of SEQ ID NO:3.
 15. The kit of claim 13, whereinsaid primers include first and second primers, and wherein: a) the basesequence of said probe consists of or is contained within the sequenceof SEQ ID NO:4; b) the base sequence of said first primer consists of oris contained within the sequence of SEQ ID NO:5; and c) the basesequence of said second primer consists of or is contained within thesequence of SEQ ID NO:6.
 16. The kit of claim 13, wherein said primersinclude first and second primers, and wherein: a) the base sequence ofsaid probe consists of or is contained within the sequence of SEQ IDNO:7; b) the base sequence of said first primer consists of or iscontained within the sequence of SEQ ID NO:8; and c) the base sequenceof said second primer consists of or is contained within the sequence ofSEQ ID NO:9.
 17. The kit of claim 13, wherein said primers include firstand second primers, and wherein: a) the base sequence of said probeconsists of or is contained within the sequence of SEQ ID NO:10; b) thebase sequence of said first primer consists of or is contained withinthe sequence of SEQ ID NO:11; and c) the base sequence of said secondprimer consists of or is contained within the sequence of SEQ ID NO 12.18. The kit of claim 13, wherein said primers includes first and secondprimers, and wherein: a) the base sequence of said probe consists of oris contained within the sequence of SEQ ID NO:13; b) the base sequenceof said first primer consists of or is contained within the sequence ofSEQ ID NO:14; and c) the base sequence of said second primer consists ofor is contained within the sequence of SEQ ID NO:15 or SEQ ID NO:16. 19.The kit of claim 13, wherein the base sequence of said probe consists ofa sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and the sequencesperfectly complementary thereto.
 20. The kit of claim 13, wherein thebase sequence of at least one of said primers consists of a sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and thesequences perfectly complementary thereto.
 21. The kit of claim 13,wherein said primers include at least two of said primers.
 22. The kitof claim 13, wherein the base sequence of each of said primers is atleast 18 bases in length.
 23. The kit of claim 22, wherein the basesequence of at least one of said primers is greater than 18 bases inlength.
 24. The kit of claim 13, wherein said probe includes adetectable label.
 25. A method of detecting whether HIV nucleic acid maybe present in a sample comprising the steps of: a) providing to saidsample an oligonucleotide probe, wherein the base sequence of said probeconsists of or is contained within a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQID NO:13, and the sequences perfectly complementary thereto, whereinsaid probe hybridizes to HIV nucleic acid in said sample to a form adetectable probe:target duplex which indicates the presence of HIVnucleic acid under hybridization conditions, and wherein said probe doesnot hybridize to human nucleic acid in said sample to form a detectableprobe non-target duplex under said conditions; and b) determiningwhether said probe hybridizes to nucleic acid present in said sample asan indication that HIV nucleic acid is present in said sample.
 26. Themethod of claim 25, the base sequence of said probe consists of or iscontained within the sequence of SEQ ID NO:1 or the complement thereof.27. The method of claim 25, wherein the base sequence of said probeconsists of or is contained within the sequence of SEQ ID NO:4 or thecomplement thereof.
 28. The method of claim 25, wherein the basesequence of said probe consists of or is contained within the sequenceof SEQ ID NO:7 or the complement thereof.
 29. The method of claim 25,wherein the base sequence of said probe consists of or is containedwithin the sequence of SEQ ID NO:10 or the complement thereof.
 30. Themethod of claim 25, herein the base sequence of said probe consists ofor is contained within the sequence of SEQ ID NO:13 or the complementthereof.
 31. The method of claim 25, further comprising the step ofamplifying a target sequence contained in HIV nucleic acid.
 32. Themethod of claim 31, wherein said amplifying is an autocatalyticprocedure performed under substantially isothermal conditions.
 33. Themethod of claim 32, wherein said amplifying is carried out in thepresence of a reverse transcriptase.
 34. The method of claim 33, whereinsaid reverse transcriptase comprises RNAse H activity.
 35. The method ofclaim 34, wherein said reverse transcriptase is the only source of RNAseH activity present during said amplifying.
 36. An oligonucleotide,wherein the base sequence of said oligonucleotide consists of or iscontained within a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID, NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, and the sequences perfectly complementary thereto, wherein saidoligonucleotide hybridizes to an HIV nucleic acid target sequence in asample to form an oligonucleotide:target duplex under hybridizationconditions, and wherein said oligonucleotide does not hybridize to humannucleic acid in said sample to form an oligonucleotide:non-target duplexunder said conditions.
 37. The oligonucleotide of claim 36, wherein saidoligonucleotide is a primer.
 38. The oligonucleotide of claim 36,wherein said oligonucleotide is a probe.
 39. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:1 or the complement thereof.
 40. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:1 or thecomplement thereof.
 41. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:2 or the complement thereof.
 42. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:2 or the complement thereof.
 43. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:3 or thecomplement thereof.
 44. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained within the sequenceof SEQ ID NO:3 or the complement thereof.
 45. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:4 or the complement thereof.
 46. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:4 or thecomplement thereof.
 47. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:5 or the complement thereof.
 48. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:5 or the complement thereof.
 49. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:6 or thecomplement thereof.
 50. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained with in the sequenceof SEQ ID NO:6 or the complement thereof.
 51. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:7 or the complement thereof.
 52. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:7 or thecomplement thereof.
 53. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:8 or the complement thereof.
 54. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:8 or the complement thereof.
 55. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:9 or thecomplement thereof.
 56. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained within the sequenceof SEQ ID NO:9 or the complement thereof.
 57. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:10 or the complement thereof.
 58. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:10 or thecomplement thereof.
 59. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:11 or the complement thereof.
 60. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:11 or the complement thereof.
 61. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:12 or thecomplement thereof.
 62. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained within the sequenceof SEQ ID NO:12 or the complement thereof.
 63. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:13 or the complement thereof.
 64. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:13 or thecomplement thereof.
 65. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:14 or the complement thereof.
 66. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:14 or the complement thereof.
 67. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:15 or thecomplement thereof.
 68. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained within the sequenceof SEQ ID NO:15 or the complement thereof.
 69. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide is containedwithin the sequence of SEQ ID NO:16 or the complement thereof.
 70. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:16 or thecomplement thereof.
 71. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained within the sequenceof SEQ ID NO:17 or the complement thereof.
 72. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:17 or the complement thereof.
 73. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:18 or thecomplement thereof.
 74. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:18 or the complement thereof.
 75. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:19 or the complement thereof.
 76. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:19 or thecomplement thereof.
 77. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is contained within the sequenceof SEQ ID NO:20 or the complement thereof.
 78. The oligonucleotide ofclaim 36, wherein the base sequence of said oligonucleotide consists ofthe sequence of SEQ ID NO:20 or the complement thereof.
 79. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is contained within the sequence of SEQ ID NO:21 or thecomplement thereof.
 80. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide consists of the sequence of SEQ IDNO:21 or the complement thereof.
 81. The oligonucleotide of claim 36,wherein the base sequence of said oligonucleotide is contained withinthe sequence of SEQ ID NO:22 or the complement thereof.
 82. Theoligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide consists of the sequence of SEQ ID NO:22 or thecomplement thereof.
 83. The oligonucleotide of claim 36, wherein thebase sequence of said oligonucleotide is at least 18 bases in length.84. The oligonucleotide of claim 36, wherein the base sequence of saidoligonucleotide is greater than 18 bases in length.
 85. Theoligonucleotide of claim 36, wherein said oligonucleotide includes adetectable label.
 86. A method of detecting HIV nucleic acid which maybe present in a sample comprising the steps of: a) providing to saidsample one or more oligonucleotide primers under conditions such thatone or more of said primers forms a complex with an HIV target nucleicacid, thereby permitting DNA synthesis to be initiated, wherein the basesequence of each of said primers consists of or is contained within asequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and thesequences perfectly complementary thereto; b) amplifying a targetsequence contained in said HIV target nucleic acid; and c) detectingamplified product from step b) as an indication of the presence HIVnucleic acid in said sample.
 87. The method of claim 86, wherein saidamplifying is an autocatalytic process which is performed undersubstantially isothermal conditions and which is carried out in thepresence of a reverse transcriptase comprising RNAse H activity, saidreverse transcriptase being the only source of RNAse H activity presentin said sample during said amplifying.
 88. The method of claim 86,wherein said detecting comprises the step of adding one or moreoligonucleotide probes to the sample, wherein the base sequence of eachsaid probe consists of or is contained with a sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:13, and the sequences perfectly complementary thereto, whereineach said probe hybridizes to said amplified product in said sample to aform a detectable probe:target complex which indicates the presence ofsaid HIV nucleic acid under hybridization conditions, and wherein saidprobe does not hybridize to human nucleic acid in said sample to form adetectable probe:non-target complex under said conditions.
 89. Themethod of claim 88, wherein said probe includes a detectable label. 90.The method of claim 86, wherein the base sequence of each of saidprimers is least 18 bases in length.
 91. The method of claim 90, whereinthe base sequence of at least one of said primers is greater than 18bases in length.
 92. The method of claim 86, wherein the base sequenceof one of said primers consists of or is contained with the sequence ofSEQ ID NO:2 or the complement thereof.
 93. The method of claim 86,wherein the base sequence of one of said primers consists of or iscontained with the sequence of SEQ ID NO:3 or the complement thereof.94. The method of claim wherein the base sequence of one of said primersconsists of or is contained with the sequence of SEQ ID NO:5 or thecomplement thereof.
 95. The method of claim 86, wherein the basesequence of one of said primers consists of or is contained with thesequence of SEQ ID NO:6 or the complement thereof.
 96. The method ofclaim 86, wherein the base sequence of one of said primers consists ofor is contained with the sequence of SEQ ID NO:8 or the complementthereof.
 97. The method of claim 86, wherein the base sequence of one ofsaid primers consists of or is contained with the sequence of SEQ IDNO:9 or the complement thereof.
 98. The method of claim 86, wherein thebase sequence of one of said primers consists of or is contained withthe sequence of SEQ ID NO:11 or the complement thereof.
 99. The methodof claim 86, wherein the base sequence of one of said primers consistsof or is contained with the sequence of SEQ ID NO:12 or the complementthereof.
 100. The method of claim 86, wherein the base sequence of oneof said primers consists of or is contained with the sequence of SEQ IDNO:14 or the complement thereof.
 101. The method of claim 86, whereinthe base sequence of one of said primers consists of or is containedwith the sequence of SEQ ID NO:15 or the complement thereof.
 102. Themethod of claim 86, wherein the base sequence of one of said primersconsists of or is contained with the sequence of SEQ ID NO:16 or thecomplement thereof.
 103. The method of claim 86, wherein the basesequence of one of said primers consists of or is contained with thesequence of SEQ ID NO:17 or the complement thereof.
 104. The method ofclaim 86, wherein the base sequence of one of said primers consists ofor is contained with the sequence of SEQ ED NO:18 or the complementthereof.
 105. The method of claim 86, wherein the base sequence of oneof said primers consists of or is contained with the sequence of SEQ IDNO:19 or the complement thereof.
 106. The method of claim 86, whereinthe base sequence of one of said primers consists of or is containedwith the sequence of SEQ ID NO:20 or the complement thereof.
 107. Themethod of claim 86, wherein the base sequence of one of said primersconsists of or is contained with the sequence of SEQ ID NO:21 or thecomplement thereof.
 108. The method of claim 86, wherein the basesequence of one of said primers consists of or is contained with thesequence of SEQ ID NO:22 or the complement thereof.
 109. The method ofclaim 86, wherein the base sequence of at least one of said primersconsists of a sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, and the sequences perfectly complementary thereto. 110.The method of claim 86, wherein said primers include at least two ofsaid primers.